Subject anti-HCV antibody detection assays employing NS3 capture peptides

ABSTRACT

The present disclosure provides methods, kits, and compositions for detecting subject anti-HCV antibodies in a sample using NS3 capture peptides. In certain embodiments, at least two NS3 helicase (NS3h) capture peptides and at least two conjugate peptides (e.g., NS3h conjugate peptides) are employed together, which allows for a broad dynamic range of subject antibody detection in a one-step type assay. In other embodiments, methods are provided of detecting NS3-specific subject antibodies without the use of a reducing agent. In some embodiments, NS3-specific subject antibodies are detected with a ‘double shot’ of NS3 conjugate peptide (e.g., conjugate peptide added to a sample both before and after washing).

The present application is a continuation of copending U.S. applicationSer. No. 16/122,261, filed Sep. 5, 2018, which is a continuation of U.S.application Ser. No. 14/925,778, filed Oct. 28, 2015, now U.S. Pat. No.10,088,483, which claims priority to U.S. Provisional Patent ApplicationNo. 62/072,266, filed Oct. 29, 2014, each of which is hereinincorporated by reference in its entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 53,106 Byte ASCII (Text) file named“34164-304_ST25.TXT,” created on Oct. 15, 2020.

FIELD OF THE INVENTION

The present disclosure provides methods, kits, and compositions fordetecting subject anti-HCV antibodies in a sample using NS3 capturepeptides. In certain embodiments, at least two NS3 helicase (NS3h)capture peptides and at least two conjugate peptides (e.g., NS3hconjugate peptides) are employed together, which allows for a broaddynamic range of subject antibody detection in a one-step type assay. Inother embodiments, methods are provided of detecting NS3-specificsubject antibodies without the use of a reducing agent. In someembodiments, NS3-specific subject antibodies are detected with a ‘doubleshot’ of NS3 conjugate peptide (e.g., conjugate peptide added to asample both before and after washing).

BACKGROUND

According to WHO statistics, as many as 170 million people worldwide areinfected by hepatitis C virus (HCV), a viral infection of the liver. 75to 85% of persons infected with HCV progress to chronic infection,approximately 20% of these cases develop complications of chronichepatitis C, including cirrhosis of the liver or hepatocellularcarcinoma after 20 years of infection. The current recommended treatmentfor HCV infections is a combination of interferon and ribavirin drugs,however the treatment is not effective in all cases and the livertransplantation is indicated in hepatitis C-related end-stage liverdisease. At present, there is no vaccine available to prevent HCVinfection, therefore all precautions to avoid infection must be taken.Therefore, sensitive HCV detection assays are important for publicsafety.

SUMMARY OF THE INVENTION

The present disclosure provides methods, kits, and compositions fordetecting subject anti-HCV antibodies in a sample using NS3 capturepeptides. In certain embodiments, at least two NS3 helicase (NS3h)capture peptides and at least two conjugate peptides (e.g., NS3hconjugate peptides) are employed together, which allows for a broaddynamic range of subject antibody detection in a one-step type assay. Inother embodiments, methods are provided of detecting NS3-specificsubject antibodies without the use of a reducing agent. In someembodiments, NS3-specific subject antibodies are detected with a ‘doubleshot’ of NS3 conjugate peptide (e.g., conjugate peptide added to asample both before and after washing).

In some embodiments, provided herein are methods of detecting hepatitisC virus (HCV) infection in a subject comprising: a) contacting aninitial biological sample (e.g., blood, plasma, serum, semen, etc.) withfirst and second NS3h (NS3 helicase) capture peptides and first andsecond detectably labeled conjugate peptides (e.g., NS3h specificconjugate peptides) to generate a mixed biological that comprises theinitial biological sample, the first and second NS3h capture peptides,and the first and second conjugate peptides, wherein the first andsecond NS3h capture peptides: i) each comprise an amino acid sequenceencoding at least one HCV NS3 helicase epitope; and ii) have differentamino acid sequences (e.g., have at least one amino acid difference,such as a difference in length or sequence), wherein the initialbiological sample is suspected of containing subject antibodies, andwherein the subject antibodies are not in purified form in thebiological sample, b) incubating the mixed biological sample underconditions such that: the first NS3h capture peptide specifically bindsat least one of the subject antibodies to form a first capture complexand the first (or second) conjugate peptide binds the subject antibodyin the first capture complex to form a first detectable complex, and thesecond NS3h capture peptide specifically binds at least one of thesubject antibodies to form a second capture complex and the second (orfirst) conjugate peptide binds the subject antibody in the secondcapture complex to form a second detectable complex; c) washing themixed biological sample to generate a washed sample; and d) detectingthe presence of the first and/or second detectable complexes, therebydetecting the presence of past or present HCV infection in the subject,wherein the presence of both the first and second NS3h capture peptidesin the mixed biological sample extends the dynamic range for detecting(e.g., qualitatively or quantitatively) the subject antibodies comparedto only using the first or second NS3h capture peptide.

In certain embodiments, the present disclosure provides kits or systemscomprising: a) first and second NS3h capture peptides, wherein the firstand second NS3h capture peptides: i) each comprise an amino acidsequence encoding at least one HCV NS3 helicase epitope; ii) havedifferent amino acid sequences, iii) are able to bind to at least onesubject antibody in a biological sample for form capture complexes, andb) first and second conjugate peptides, wherein the first and secondconjugate peptides are able to bind to the subject antibodies in thecapture complexes. In certain embodiments, the kits and systems furthercomprise: c) the biological sample containing the subject antibodies,and wherein the subject antibodies are not in purified form in thebiological sample.

In further embodiments, provided herein are compositions comprising: a)first and second NS3h capture peptides, wherein the first and secondNS3h capture peptides: i) each comprise an amino acid sequence encodingat least one HCV NS3 helicase epitope; ii) have different amino acidsequences, iii) are able to bind to at least one subject antibody in abiological sample for form capture complexes, and b) first and secondconjugate peptides, wherein the first and second conjugate peptides areable to bind to the subject antibodies in the capture complexes. Incertain embodiments, compositions further comprise c) the biologicalsample containing the subject antibodies, and wherein the subjectantibodies are not in purified form in the biological sample. In otherembodiments, the compositions are free or detectably free of detergents.

In particular embodiments, provided herein are methods of detectinghepatitis C virus (HCV) infection in a subject comprising: a) contactinga sample suspected of containing subject antibodies with: i) a firstNS3h capture peptide comprising an amino acid sequence with at least 80%or 90% sequence identity (e.g., at least 80% . . . 90% . . . 95% . . .98% . . . 99.5% . . . or 100% sequence identity) with Domain 1 and/orDomain 2 of an HCV NS3 helicase, wherein the first NS3h capture peptideis no more than 400 or 350 or 300 amino acids in length (e.g., no morethan 400 . . . 375 . . . 325 . . . 300 . . . 275 . . . 250 . . . 225 . .. 200 . . . or 180 amino acids in length); ii) a second NS3h capturepeptide which comprises an amino acid sequence with at least 85% or 90%or 95% (e.g., at least 85% . . . 90% . . . 94% . . . 97% . . . 99% . . .99.5% . . . 99.9% . . . or 100%) sequence identity with a full-lengthHCV NS3 helicase that comprises Domains 1, 2, and 3 of an HCV NS3helicase, and b) incubating the sample under conditions such that thefirst NS3h capture peptide specifically binds at least one of thesubject antibodies to form a first complex, and the second NS3h capturepeptide specifically binds at least one of the subject antibodies toform a second complex; and c) detecting the presence of the first and/orsecond complex, thereby detecting the presence of past or present HCVinfection in the subject. In particular embodiments, the presence of thefirst NS3h capture peptide along with the second NS3h capture peptide inthe sample extends the upper dynamic range for qualitatively detectingthe subject antibodies compared to only using the second NS3h peptide.

In certain embodiments, provided herein are kits and systems comprising:a) a first NS3h capture peptide comprising an amino acid sequence withat least 80% or 90% sequence identity with Domain 1 and/or Domain 2 ofan HCV NS3 helicase, wherein the first NS3h peptide is no more than 400or 350 or 300 or 225 amino acids in length; b) a second NS3h capturepeptide comprising an amino acid sequence with at least 85% or 95%sequence identity with a full-length HCV NS3 helicase that comprisesDomains 1, 2, and 3 of an HCV NS3 helicase.

In certain embodiments, the kits and systems further comprise a firstcontainer, wherein the first and second NS3h capture peptides are insidethe first container. In other embodiments, the first container is freeor substantially free of a detergent. In other embodiments, the kits andsystems further comprise a solid support (e.g., microparticles). Inother embodiments, the kits and systems further comprise a secondcontainer, wherein the solid support is inside the second container. Infurther embodiments, the kits and systems further comprise a firstdetectably labeled conjugate peptide (e.g., that will bind to a subjectantibody captured by the first or second NS3h capture peptide). Incertain embodiments, the first detectably labeled conjugate peptide: i)comprises an amino acid sequence with at least 80% or 90% sequenceidentity with Domain 1 of an HCV NS3 helicase, ii) is no more than 250or 200 amino acids in length; and iii) comprises a detectable label. Inadditional embodiments, the kits and systems further comprise a seconddetectably labeled conjugate peptide (e.g., that will bind to a subjectantibody captured by the first or second NS3h capture peptide). Incertain embodiments, the second detectably labeled conjugate peptide: i)comprises an amino acid sequence with at least 80% or 90% sequenceidentity with a full-length HCV NS3 helicase that has Domains 1, 2, and3 of an HCV NS3 helicase, and ii) comprises a detectable label. In someembodiments, the kits and systems further comprise a second (or third)container, wherein the first and second NS3h conjugate peptides areinside the second (or third) container. In particular embodiments, thesecond container is free or substantially free of detergent.

In certain embodiments, the kits and systems further comprise a firstanti-HCV antibody. In some embodiments, the first anti-HCV antibody isan anti-core HCV antibody or anti-NS3 or NS4 antibody. In otherembodiments, the first anti-HCV antibody comprises a solid-supportbinding moiety. In further embodiments, the kits and systems furthercomprises a second anti-HCV antibody. In certain embodiments, the secondanti-HCV antibody is an anti-core HCV antibody. In some embodiments, thekits and systems further comprise a second container, wherein the secondanti-HCV antibody is inside the second container. In certainembodiments, the kits and systems further comprise an HCV core capturepeptide. In additional embodiments, the HCV core capture peptidecomprises a solid-support binding moiety. In other embodiments, the kitsand systems further comprise an HCV detectably labeled conjugate corepeptide.

In certain embodiments, the present disclosure provides compositionscomprising: a) a first NS3h capture peptide comprising an amino acidsequence with at least 80% or 90% (e.g., 80% . . . 90% . . . 95% . . .99% . . . 99.5%) sequence identity with Domain 1 and/or Domain 2 of anHCV NS3 helicase, wherein the first NS3h peptide is no more than 250 or350 amino acids in length (e.g., no more than 250 . . . 300 . . . or350); and b) a second NS3h capture peptide comprising an amino acidsequence with at least 90% or 95% sequence identity with a full-lengthHCV NS3 helicase that comprises Domains 1, 2, and 3 of an HCV NS3helicase. In some embodiments, the compositions further comprise atleast one of the following components: c) a solid support; d) a firstdetectably labeled conjugate peptide; e) a second detectably labeledconjugate peptide; f) a first anti-HCV antibody; g) a second anti-HCVantibody which is detectably labeled; h) an HCV core capture peptide;and i) a detectably labeled HCV core conjugate peptide. In particularembodiments, the composition has at least two, three, four, five, six,or all seven of the components.

In certain embodiments, provided herein are compositions comprising atleast one peptide comprising or consisting of the amino acid sequence inany one of SEQ ID NOs:1-16, or a peptide with at least 95% (e.g., 95% .. . 98% . . . 99% . . . or 99.5%) sequence identity with any one of SEQID NOs:1-16.

In some embodiments, provided herein are methods of detecting hepatitisC virus (HCV) infection in a subject comprising: a) contacting a samplesuspected of containing subject antibodies with an NS3 capture peptide,wherein the NS3 capture peptide comprises an amino acid sequenceencoding at least one HCV NS3 epitope, and wherein the contacting isconducted under conditions such that no exogenous reducing agent (i.e.,a reducing agent not naturally present in the sample) is added to, orpresent in, the sample, b) incubating the sample under conditions suchthat the NS3 capture peptide specifically binds at least one of thesubject antibodies to form a first complex, wherein the incubating isconducted under conditions wherein no exogenous reducing agent ispresent in the sample; and c) detecting the presence of the firstcomplex, thereby detecting the presence of past or present HCV infectionin the subject, wherein the detecting is conducted under conditionwherein no exogenous reducing agent is present.

In certain embodiments, provided herein are kits, systems, andcompositions comprising: a composition comprising a biological sample,an NS3 capture peptide, a detectably labeled conjugate peptide, and aplurality of subject antibodies, wherein the NS3 capture peptide and theconjugate peptide are bound to at least one subject antibody, andwherein the composition is free from exogenous reducing agents.

In some embodiments, provided herein are methods of detecting hepatitisC virus (HCV) infection in a subject comprising: a) contacting aninitial biological sample with a first NS3 capture peptide and a firstconjugate peptide to generate a mixed biological that comprises theinitial biological sample, the first NS3 capture peptide and the firstconjugate peptides, wherein the NS3 capture peptide comprises an aminoacid sequence encoding at least one HCV NS3 helicase epitope; whereinthe initial biological sample is suspected of containing subjectantibodies, and wherein the subject antibodies are not in purified formin the biological sample, b) incubating the mixed biological sampleunder conditions such that the NS3 capture peptide specifically binds atleast one of the subject antibodies to form a capture complex and theconjugate peptide binds the subject antibody in the capture complex toform a detectable complex, and c) washing the mixed biological sample togenerate a washed sample; d) adding additional amounts of the conjugatepeptide or a different conjugate peptide that specifically binds thesubject antibody in the capture complex; and e) detecting the presenceof the detectable complex, thereby detecting the presence of past orpresent HCV infection in the subject. In further embodiments, theconjugate peptide binds to at least one epitope of NS3h (NS3h helicaseof HCV).

In certain embodiments, the amino acid sequence of the second NS3hcapture peptide is at least 1.5 or 2 times longer than the first NS3hcapture peptide (e.g., at least 1.5 . . . 3.0 . . . 4.0 . . . 5.0 timeslonger or more). In some embodiments, the first and second conjugateepitopes are specific for an NS3h epitope. In further embodiments, thefirst NS3h capture peptide has at least 80% or 90% (e.g., 80% . . . 87%. . . 94% . . . 98% . . . 99%, . . . or 95.5%) sequence identity with anHCV NS3 helicase Domain 1, and is no more than 250 amino acids inlength. In further embodiments, the first NS3h capture peptide has atleast 80 or 90% sequence identity with an HCV NS3 helicase Domain 2, andis no more than 250 amino acids in length (e.g., no more than 250 . . .225 . . . 200 . . . or 180 amino acids in length). In additionalembodiments, the second NS3h capture peptide has at least 90% or 95%(e.g., 90% . . . 94% . . . 96% . . . or 99%) sequence identity with afull-length NS3 helicase having Domains 1, 2, and 3. In certainembodiments, the second NS3h capture peptide comprises a full-length NS3helicase sequence having Domains 1, 2, and 3. In further embodiments,the amino acid sequence of the second NS3h capture peptide has at least99% sequence identity with the full-length HCV helicase. In additionalembodiments, the amino acid sequence of the second NS3h capture peptidecomprises a full-length NS3 helicase sequence having Domains 1, 2, and3. In further embodiments, the second NS3h capture peptide has a NS3helicase native structure (i.e., not denatured structure).

In some embodiments, the sample is further suspected of containing HCVparticles or fragments thereof, and wherein the method further comprisescontacting the sample with a first anti-HCV capture antibody such that athird complex is formed, wherein the third complex comprises the firstanti-HCV capture antibody bound to an HCV particle or fragment thereof.In other embodiments, the first capture anti-HCV antibody is ananti-core HCV antibody. In additional embodiment, the first anti-HCVantibody comprises a solid-support binding moiety. In additionalembodiments, the methods further comprise contacting the sample with asecond anti-HCV antibody (e.g., conjugate antibody) that binds the HCVparticle or fragment thereof in the third complex, wherein the secondanti-HCV antibody is detectably labeled. In further embodiments, the thesecond anti-HCV antibody is an anti-core HCV antibody. In someembodiments, the methods further comprise detecting the third complex.

In certain embodiments, prior to any detection by conjugate peptide orconjugate antibody, the sample is washed. In some embodiments, themethods further comprise contacting the sample with a first HCV corepeptide (e.g., core capture peptide), wherein the first core peptidespecifically binds at least one of the subject antibodies to form afourth complex. In additional embodiments, the first HCV core peptidecomprises or consists of the amino acid sequence shown in SEQ ID NO:12or one with 95% or more identity with SEQ ID NO:12. In furtherembodiments, the first HCV core peptide comprises a solid-supportbinding moiety.

In additional embodiments, provided the methods further comprisecontacting the sample with a second HCV core peptide (e.g., conjugatepeptide), wherein the second HCV core peptide is detectably labeled, andwherein the second HCV core peptide binds to the subject antibody aspart of the fourth complex. In certain embodiments, the methods furthercomprise detecting the presence of the fourth complex. In additionalembodiments, the methods further comprise contacting the sample with asolid support (e.g., microbeads, etc.). In further embodiments, thesolid support is coated with avidin or other binding molecule.

In additional embodiments, the methods further comprise contacting thesample with a first detectably labeled conjugate peptide that binds tothe subject antibody as part of the first complex, and wherein thedetecting the presence of the first complex comprises detecting thefirst detectably labeled conjugate peptide. In further embodiments, thefirst detectably labeled conjugate peptide: i) comprises an amino acidsequence with at least 80% . . . 90% . . . or 99% sequence identity withDomain 1 of an HCV NS3 helicase, ii) is no more than 300 . . . 200 . . .or 180 amino acids in length; and iii) comprises a detectable label.

In certain embodiments, the methods further comprise contacting thesample with a second detectably labeled conjugate peptide that binds tothe subject antibody as part of the second complex, and wherein thedetecting the presence of the second complex comprises detecting thesecond detectably labeled conjugate peptide. In other embodiments, thesecond detectably labeled conjugate peptide comprises an amino acidsequence with at least 80% . . . 90% . . . 99% sequence identity withthe full-length HCV NS3 helicase. In additional embodiments, the firstNS3h capture peptide is no more than 180 amino acids in length. Infurther embodiments, the first NS3h capture peptide comprises orconsists of the amino acid sequence in SEQ ID NO:2 or SEQ ID NO:3, orwherein the first NS3 peptide has 90% . . . or 95% identity with SEQ IDNO:2 or SEQ ID NO:3. In additional embodiments, the first NS3h capturepeptide comprises or consists of at least 100 contiguous amino acids(e.g., at least 100 . . . 125 . . . or 135) from SEQ ID NO:2 or SEQ IDNO:3.

In further embodiments, the second NS3h capture peptide has at least 90%. . . or 95% sequence identity with the full-length HCV NS3 helicase. Incertain embodiments, the second NS3h capture peptide has at least 99%sequence identity with the full-length HCV NS3 helicase. In otherembodiments, the second NS3h capture peptide comprises or consists ofthe amino acid sequence in SEQ ID NO:5 or SEQ ID NO:7, or wherein thesecond NS3h capture peptide has 90% . . . or 95% sequence identity withSEQ ID NO:5 or SEQ ID NO:7. In additional embodiments, the second NS3hcapture peptide comprises or consists of at least 300 . . . 350contiguous amino acids from SEQ ID NO:5 or SEQ ID NO:7.

In further embodiments, the first conjugate peptide has at least 90% . .. or 95% identity with Domain 1 of an HCV NS3 helicase. In otherembodiments, the first conjugate peptide is no more than 180 amino acidsin length. In other embodiments, the first conjugate peptide comprisesor consists of the amino acid sequence in SEQ ID NO:2 or SEQ ID NO:3, orwherein the first conjugate peptide has 95% identity with SEQ ID NO:2 orSEQ ID NO:3. In additional embodiments, the second conjugate peptide hasat least 95% sequence identity with the full-length HCV NS3 helicase. Inother embodiments, the second conjugate peptide has at least 99%sequence identity with the full-length HCV NS3 helicase. In certainembodiments, the second conjugate peptide comprises or consists of theamino acid sequence in SEQ ID NO:5 or SEQ ID NO:7, or wherein the secondconjugate peptide has 95% identity with SEQ ID NO:5 or SEQ ID NO:7.

In some embodiments, the Domain 1 of an HCV NS3 helicase is from a HCVgenotype selected from the group consisting of: 1a, 1b, 2a, 2b, 2c, 2d,3a, 3b, 3c, 3d, 3e, 3f, 4a, 4b, 4c, 4d, 4e, 4f, 4g, 4h, 4i, 4j, 5a, and6a. In further embodiments, the full-length HCV NS3 helicase is from aHCV genotype selected from the group consisting of: 1a, 1 b, 2a, 2b, 2c,2d, 3a, 3b, 3c, 3d, 3e, 3f, 4a, 4b, 4c, 4d, 4e, 4f, 4g, 4h, 4i, 4j, 5a,and 6a. In other embodiments, the subject is a human. In additionalembodiments, the solid-support binding moiety comprises biotin. Infurther embodiments, methods further comprise adding a trigger solutionto the sample, wherein the trigger solution triggers a signal from thedetectable label.

DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the amino acid sequences of exemplary peptidetermed NS3-D1, which include amino acids 1192-1356 of HCV according tothe standard numbering, which includes all of the helicase Domain 1(1207-1356), and a small portion of the NS3 protease (119201207). FIG.1A specifically shows the amino acid sequences of NS3-D1 capture peptide(SEQ ID NO:2) with an N-terminal histidine tag (e.g., for proteinpurification), while FIG. 1B shows a biotinylated version of NS3-D1 (SEQID NO:3) (e.g., such that this peptide can bind to avidin-coatedmicrobeads). FIGS. 1C-E show an exemplary sequence termed “NS3-D1,DelN15,” which is amino acids 1205-1356. FIG. 1C specifically shows theamino acid sequence of exemplary peptide NS3-D1, delN15-Cbt (v2e), SEQID NO:1, with a biotin sequence at the C-terminus. FIG. 1D specificallyshows the amino acid sequence of exemplary peptide NS3-D1, delN15-XC9,SEQ ID NO:13, which has the “XC9” sequence at the C-terminal end, whichis designed to bind acridinylated BSA for labeling purposes. FIG. 1Eprovides the amino acid sequence of NS3-D1, del N15 (SEQ ID NO:14), withan N-terminal histidine tag.

FIGS. 2A-2F show the nucleic acid and amino acid sequences of exemplaryNS3h peptides NS3-(DeltaN15) (amino acids 1205-1658) which includes thefull-length HCV NS3 helicase with all three domains. FIG. 2Aspecifically shows the nucleic acid sequence of NS3h(deltaN15) (SEQ IDNO:4). FIG. 2B shows the amino acid sequence of NS3h(deltaN15) (SEQ IDNO:5). FIG. 2C shows the nucleic acid sequence ofNS3h(deltaN15)-Cbt(v2e) (SEQ ID NO:6), which encodes a c-terminal biotintag. FIG. 2D shows the amino acid sequence of NS3h(deltaN15)-Cbt(v2e)(SEQ ID NO:7), which includes a c-terminal biotin tag. FIG. 2E shows thenucleic acid sequence of NS3h(deltaN15)-XC9 (SEQ ID NO:8), which encodesa c-terminal XC9 sequence. FIG. 2F shows the amino acid sequence ofNS3h(deltaN15)-XC9 (SEQ ID NO:9), which encodes an XC9 sequence designedto bind acridinylated BSA for labeling purposes.

FIG. 3A shows the nucleic acid sequence (SEQ ID NO:10) of the fusionpeptide 9NB45H, and FIG. 3B shows the amino acid sequence (SEQ ID NO:11)of fusion peptide 9NB45H, which is a fusion of amino acids 1192-1457(NS3 region), 1-150 (core region), and GSGSHHHHHH (histidine tag). Thissequence is useful, for example, as a calibrator or control sequence.

FIG. 4 shows the amino acid sequence of an exemplary core peptide (SEQID NO:12), which is amino acids 15-68, with a deletion at positions 34and 48.

FIG. 5 shows a sequence alignment of the sequences described in FIG. 1-4and the isolate P26664 in the NS3 region of HCV. This alignment shows aportion of the protease, as well as the location of domains 1, 2, and 3of the HS3 helicase.

FIG. 6A shows the amino acid sequence of peptide 33c (aka 9NB49), whichis amino acids 1192-1457, and FIG. 6B shows the corresponding nucleicacid sequence.

DEFINITIONS

The term “sample,” as used herein, is used in its broadest sense. A“biological sample”, as used herein, includes, but is not limited to,any quantity of a substance from a living thing or formerly livingthing. Such living things include, but are not limited to, humans, mice,rats, monkeys, dogs, rabbits and other animals. Such substances include,but are not limited to, blood, (e.g., whole blood or componentsthereof), plasma, serum, urine, saliva, amniotic fluid, synovial fluid,endothelial cells, leukocytes, monocytes, other cells, organs, tissues,bone marrow, lymph nodes and spleen.

The term “antibody” (Ab) and “antibodies” (Abs) refer to monoclonalantibodies (mAb (singular) or mAbs (plural)), polyclonal antibodies(pAbs (plural)), multispecific antibodies, human antibodies, humanizedantibodies (fully or partially humanized; a polypeptide comprising amodified variable region of a human antibody wherein a portion of thevariable region has been substituted by the corresponding sequence froma non-human sequence and wherein the modified variable region is linkedto at least part of the constant region of a human antibody), animalantibodies (such as, but not limited to, a bird (for example, a duck ora goose), a shark, a whale, and a mammal, including a non-primate (forexample, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, asheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, etc.) or anon-human primate (for example, a monkey, a chimpanzee, etc.),recombinant antibodies, chimeric antibodies (cAb; a polypeptidecomprising all or a part of the heavy and light chain variable regionsof an antibody from one host species linked to at least part of theantibody constant regions from another host species), single chainantibodies, single domain antibodies, Fab fragments, F(ab′) fragments,Fab′-SH fragments, F(ab′)2 fragments, Fd fragments, Fv fragments,single-chain Fv fragments (“scFv”), disulfide-linked Fv fragments(“sdFv”), dAb fragments, diabodies, an isolated complementaritydetermining region (CDR), and anti-idiotypic (“anti-Id”) antibodies,bifunctional or dual-domain antibodies (e.g., dual variable domainantibodies, or DVD-IgGs), and functionally active, epitope-bindingfragments (or antigenically reactive fragments) of any of the above. Inparticular, antibodies include immunoglobulin molecules andimmunologically active (or antigenically reactive) fragments ofimmunoglobulin molecules, namely, molecules that contain ananalyte-binding site as further described in (n) herein, and variants asfurther described herein. Immunoglobulin molecules can be of any type(for example, IgG, IgE, IgM, IgD, IgA and IgY), class (for example,IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or subclass. An antibody, whoseaffinity (namely, KD, kd or ka) has been increased or improved via thescreening of a combinatory antibody library that has been prepared usingbio-display, is referred to as an “affinity maturated antibody.” Forsimplicity sake, an antibody against an analyte is frequently referredto herein as being either an “anti-analyte antibody” or merely an“analyte antibody” (e.g., an anti-HCV antibody or an HCV antibody).

In the present description, the assay “component,” “components,” and “atleast one component,” refer to, for example, a capture antibodies,capture peptides (e.g., first and second NS3h capture antibodies), adetection or conjugate antibody, a control, a calibrator, a series ofcalibrators, a sensitivity panel, a container, a buffer, a diluent, asalt, an enzyme, a co-factor for an enzyme, a detection reagent, apretreatment reagent/solution, a substrate (e.g., as a solution), a stopsolution, and the like that can be included in a kit for assay of a testsample, such as a patient urine, serum or plasma sample, in accordancewith the methods described herein and other methods known in the art.Thus, in the context of the present disclosure, “at least onecomponent,” “component,” and “components” can include a polypeptide asdescribed herein, which is optionally immobilized on a solid support.Some components can be in solution or lyophilized for reconstitution foruse in an assay.

In conducting the assays of the present disclosure, it may be useful touse a control. “Control” refers to a composition known to not containanti-HCV antibody (“negative control”) or to contain anti-HCV antibody(“positive control”). A positive control can comprise a knownconcentration of anti-HCV antibody. “Control,” “positive control,” and“calibrator” may be used interchangeably herein to refer to acomposition comprising a known concentration of anti-HCV antibody. A“positive control” can be used to establish assay performancecharacteristics and is a useful indicator of the integrity of reagents(e.g., analytes).

“Epitope,” “epitopes” and “epitopes of interest” refer to a site(s) onany molecule (e.g., the NS3 antigens described herein) that isrecognized and can bind to a complementary site on a specific bindingpartner, such as an antibody or antigenically reactive fragment thereof.An epitope is composed of the precise amino acid residues of a region ofan antigen (or fragment thereof) known to bind to the complementary siteon the specific binding partner. An antigenic fragment can contain morethan one epitope. In the assays, kits, and compositions that aredescribed herein, one or other component of the assay may comprise adetectable label. The terms “label” and “detectable label” mean a moietyattached to a specific binding partner, such as an antibody or ananalyte, to render the reaction between members of a specific bindingpair, such as an antibody and an analyte, detectable, and the specificbinding partner, e.g., antibody or analyte, so labeled is referred to as“detectably labeled.” A label can produce a signal that is detectable byvisual or instrumental means. Various labels include signal-producingsubstances, such as chromogens, fluorescent compounds, chemiluminescentcompounds, radioactive compounds, and the like. Representative examplesof labels include moieties that produce light, e.g., acridiniumcompounds, and moieties that produce fluorescence, e.g., fluorescein.Other labels are described herein. In this regard, the moiety itself maynot be detectably labeled but may become detectable upon reaction withyet another moiety. Use of “detectably labeled” is intended to encompassthe latter type of detectable labeling.

“Linking sequence” refers to a natural or artificial polypeptidesequence that is connected to one or more polypeptide sequences ofinterest (e.g., full-length, fragments, etc.). The term “connected”refers to the joining of the linking sequence to the polypeptidesequence of interest. Such polypeptide sequences are preferably joinedby one or more peptide bonds. Linking sequences can have a length offrom about 4 to about 50 amino acids. Preferably, the length of thelinking sequence is from about 6 to about 30 amino acids. Naturallinking sequences can be modified by amino acid substitutions,additions, or deletions to create artificial linking sequences.Exemplary linking sequences include, but are not limited to: (i)Histidine residues (His tags), such as a 6× His tag, which contains sixhistidine residues, are useful as linking sequences to facilitate theisolation and purification of polypeptides and antibodies of interest.(ii) Enterokinase cleavage sites, like His tags, are used in theisolation and purification of proteins and antibodies of interest.Often, enterokinase cleavage sites are used together with His tags inthe isolation and purification of proteins and antibodies of interest.Various enterokinase cleavage sites are known in the art. (iii)Miscellaneous sequences can be used to link or connect the light and/orheavy chain variable regions of single chain variable region fragments.Examples of other linking sequences can be found in Bird et al., Science242: 423-426 (1988); Huston et al., PNAS USA 85: 5879-5883 (1988); andMcCafferty et al., Nature 348: 552-554 (1990). Linking sequences alsocan be modified for additional functions, such as attachment of drugs orattachment to solid supports. In the context of the present disclosure,an mAb, for example, can contain a linking sequence, such as a His tag,an enterokinase cleavage site, or both.

“Patient” and “subject” may be used interchangeably herein to refer toan animal, such as a bird (e.g., a duck or a goose), a shark, a whale,and a mammal, including a non-primate (for example, a cow, a pig, acamel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guineapig, a cat, a dog, a rat, and a mouse) and a primate (for example, amonkey, a chimpanzee, and a human). Preferably, the patient or subjectis a human, such as a human at risk for HCV infection or a humaninfected with HCV.

In analysis of the results of the immunoassays described herein it maybe useful to include certain levels of detection as cutoff levels.“Predetermined cutoff” and “predetermined level” refer generally to anassay cutoff value that is used to assessdiagnostic/prognostic/therapeutic efficacy results by comparing theassay results against the predetermined cutoff/level, where thepredetermined cutoff/level already has been linked or associated withvarious clinical parameters (e.g., severity of disease,progression/nonprogression/improvement, etc.). While the presentdisclosure may provide exemplary predetermined levels, it is well-knownthat cutoff values may vary depending on the nature of the immunoassay(e.g., antibodies employed, etc.). It further is well within theordinary skill of one in the art to adapt the disclosure herein forother immunoassays to obtain immunoassay-specific cutoff values forthose other immunoassays based on this disclosure. Whereas the precisevalue of the predetermined cutoff/level may vary between assays, thecorrelations as described herein should be generally applicable.

As described below, it may be desirable in some embodiments to provide apretreatment of the test sample. “Pretreatment reagent,” e.g., lysis,precipitation and/or solubilization reagent, as used in a diagnosticassay as described herein is one that lyses any cells and/or solubilizesany analyte that is/are present in a test sample. Pretreatment is notnecessary for all samples, as described further herein. Among otherthings, solubilizing the analyte (i.e., anti-HCV antibody) entailsrelease of the analyte from any endogenous binding proteins present inthe sample. A pretreatment reagent may be homogeneous (not requiring aseparation step) or heterogeneous (requiring a separation step). Withuse of a heterogeneous pretreatment reagent there is removal of anyprecipitated analyte binding proteins from the test sample prior toproceeding to the next step of the assay. The pretreatment reagentoptionally can comprise: (a) one or more solvents and salt, (b) one ormore solvents, salt and detergent, (c) detergent, (d) detergent andsalt, or (e) any reagent or combination of reagents appropriate for celllysis and/or solubilization of analyte.

The assays also may be subject to rigorous quality control. “Qualitycontrol reagents” in the context of immunoassays and kits describedherein, include, but are not limited to, calibrators, controls, andsensitivity panels. A “calibrator” or “standard” typically is used(e.g., one or more, such as a plurality) in order to establishcalibration (standard) curves for interpolation of the concentration ofan analyte, such as an antibody or an analyte. Alternatively, a singlecalibrator, which is near a predetermined positive/negative cutoff, canbe used. Multiple calibrators (i.e., more than one calibrator or avarying amount of calibrator(s)) can be used in conjunction so as tocomprise a “sensitivity panel.”

The terms “sample,” “test sample,” and “patient sample” may be usedinterchangeably herein. The sample, such as a sample of urine, serum,plasma, amniotic fluid, cerebrospinal fluid, placental cells or tissue,endothelial cells, leukocytes, or monocytes, can be used directly asobtained from a patient or can be pre-treated, such as by filtration,distillation, extraction, concentration, centrifugation, inactivation ofinterfering components, addition of reagents, and the like, to modifythe character of the sample in some manner as discussed herein orotherwise as is known in the art. Preferably, the sample is urine, serumor plasma.

In some assays, it may be desirable to provide calibration of the assay.“Series of calibrating compositions” refers to a plurality ofcompositions comprising a known concentration of anti-HCV antibody,wherein each of the compositions differs from the other compositions inthe series by the concentration of anti-HCV antibody.

Throughout the present specification, it is noted that the NS3h antigensand/or other reagents may be bound to a solid support or solid phase,both of which terms are used interchangeably. The term “solid phase”refers to any material that is insoluble, or can be made insoluble by asubsequent reaction. The solid phase can be chosen for its intrinsicability to attract and immobilize a capture agent. Alternatively, thesolid phase can have affixed thereto a linking agent that has theability to attract and immobilize the capture agent. The linking agentcan, for example, include a charged substance that is oppositely chargedwith respect to the capture agent itself or to a charged substanceconjugated to the capture agent. In general, the linking agent can beany binding partner (preferably specific) that is immobilized on(attached to) the solid phase and that has the ability to immobilize thecapture agent through a binding reaction. The linking agent enables theindirect binding of the capture agent to a solid phase material beforethe performance of the assay or during the performance of the assay(e.g., capture on the fly type assays). The solid phase can, forexample, be plastic, derivatized plastic, magnetic or non-magneticmetal, glass or silicon, including, for example, a test tube, microtiterwell, sheet, beads, microparticles, chip, and other configurations knownto those of ordinary skill in the art.

In certain descriptions of the assays, kits, and compositions describedherein, it may be useful to refer to either the NS3, NS4 or core antigenor the HCV antibody as a specific binding partner. “Specific bindingpartner” is a member of a specific binding pair. A specific binding paircomprises two different molecules, which specifically bind to each otherthrough chemical or physical means. Therefore, in addition to antigenand antibody specific binding pairs of common immunoassays, otherspecific binding pairs can include biotin and avidin (or streptavidin),carbohydrates and lectins, complementary nucleotide sequences, effectorand receptor molecules, cofactors and enzymes, enzyme inhibitors andenzymes, and the like. Furthermore, specific binding pairs can includemembers that are analogs of the original specific binding members, forexample, an analyte-analog. Immunoreactive specific binding membersinclude antigens, antigen fragments, and antibodies, includingmonoclonal and polyclonal antibodies as well as complexes, fragments,and variants (including fragments of variants) thereof, whether isolatedor recombinantly produced. The term “specific” and “specificity” in thecontext of an interaction between members of a specific binding pair(e.g., an antigen (or fragment thereof) and an antibody (orantigenically reactive fragment thereof)) refer to the selectivereactivity of the interaction. The phrase “specifically binds to” andanalogous phrases refer to the ability of antibodies (or antigenicallyreactive fragments thereof) to bind specifically to a given antigen (ora fragment thereof) and not bind specifically to other entities.

DETAILED DESCRIPTION

The present disclosure provides methods, kits, and compositions fordetecting subject anti-HCV antibodies in a sample using NS3 capturepeptides. In certain embodiments, at least two NS3 helicase (NS3h)capture peptides and at least two conjugate peptides (e.g., NS3hconjugate peptides) are employed together, which allows for a broaddynamic range of subject antibody detection in a one-step type assay. Inother embodiments, methods are provided of detecting NS3-specificsubject antibodies without the use of a reducing agent. In someembodiments, NS3-specific subject antibodies are detected with a ‘doubleshot’ of NS3 conjugate peptide (e.g., conjugate peptide added to asample both before and after washing).

In certain embodiments, the first NS3h capture peptide is composed ofsubstantially only domain 1 of a Hepicavirus (e.g., HCV) NS3 helicaseprotein (e.g., amino acids 1192-1356 or 1205-1356), while the secondNS3h capture peptide is composed of substantially all three domains ofthe NS3 helicase protein (e.g., amino acids 1207-1654). These two typesof proteins may then be added at appropriate levels in a 1-stepimmunoassay format (both capture and conjugate peptides added at, orabout, the same time to a sample prior to any wash) such that samplescontaining high titer HCV anti-NS3 antibodies are detected by the domain1 NS3 capture protein and low titer Ab samples are detected by thefull-length NS3 helicase peptide. In some embodiments, the first andsecond NS3h capture peptides contain biotin at the c-terminus (e.g., tobind to avidin coated beads), and a second set of similar or identicalproteins are used as conjugate proteins to detect captured antibodies.Such conjugate peptides may be conjugated to acridinylated BSA. Incertain embodiments, these four peptides (two capture and two conjugate)are used in a one-step type immunoassay for the detection of antibodiesto the HCV NS3 protein.

As shown in Example 1 below, the use of two types of NS3h capturepeptides allows for the dynamic range of a 1-step immunoassay for thedetection of antibodies to be extended when the two NS3h captureproteins are used in concert. For example, the NS3h domain 1 proteindetects samples containing higher concentrations of antibody and thefull-length helicase protein detects samples containing lowerconcentrations of antibody.

In general, 1-step immunoassays tend to have better sensitivity andprecision than 2-step assays but the shortfall of the 1-step format is“hook effect.” Samples containing high concentrations of analyte in thepatient sample overcome the inputs of capture and detection moleculesresulting in decreased assay response giving inaccurately low results.In a qualitative assay the decreased result can go below the cutoffresulting in a false negative result. The present disclosure, in certainembodiments, resolves the problem of “hook effect” in a 1-step assay forthe detection of antibodies. The present disclosure improves upon whatis know, for example, by extending the upper dynamic range of antibodydetection by, for example, adding a 2nd protein of lower molecularweight. In particular embodiments, this also simplifies the assayconfiguration improving the stability in an HCV Ag/Ab Combo format.

In certain embodiments, the methods, kits, and compositions of thepresent description employ at least first and/or second NS3h captureantibodies. In some embodiments, the NS3h capture peptides have an aminoacid sequence comprising or consisting of those shown in FIGS. 1-2 and5. Variants of these peptides may also be employed that include longer,shorter, or mutated versions of such amino acid sequences (e.g.,sequences with 75% . . . 85% . . . 95% . . . or 99% sequence identitywith the sequences in these figures or variants thereof). In certainembodiments, the first NS3h capture peptide is composed mainly of onlyHCV NS3h domain 1, or domain 2, or both domains 1 and 2. It is notedthat the generally accepted boundaries of the HCV NS3 helicase are asfollows: NS3 helicase Domain 1—aprox. 1207-1356 (181-330); NS3 helicaseDomain 2—aprox. 1357-1507 (331-481); and

NS3 helicase Domain 3—aprox. 1508-1654 (482-626). In certainembodiments, additional NS3 peptides or variants thereof are provided inU.S. Publ. 20140272933 entitled “HCV Antigen-Antibody Combination Assayand Methods and Compositions for use therein” and U.S. Publication20140272932 entitled “HCV NS3 Recombinant Antigens and Mutants Thereoffor Improved Antibody Detection,” both of which are specificallyincorporated by reference herein in their entireties, including for theNS3 peptide sequences disclosed therein.

In certain embodiments, the assays described herein further detect thepresence of antibodies to Core antigen. Some exemplary core antigensthat could be used include antigens derived from the DNA binding domain(amino acids 1-125) of core protein. Still other preferred core antigensare derived from the lipid binding domain of core located at amino acidresidues 134-171 of core protein(MGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG; SEQ ID NO:17) or from positions15-68, without or without deletions or other changes (see SEQ ID NO:12,which has deletions at 34 and 48). Thus, the core antigens can be coatedonto a solid phase support and/or used in solution phase to captureantibodies present in human serum or plasma that are directed toward theCore region of HCV. Preferably, such core antigens can evade detectionby the conjugate antibody used for the detection of Core antigen presentin a test sample in an HCV combination immunoassay. Thus a combinationimmunoassay can be performed that detects both Core antigen present inthe test sample at the same time as detecting anti-Core antibodies thatwould also be expected to be in the test sample and identified in thesame HCV Combo assay format.

As noted herein throughout the methods of the present disclosure aregenerally immunoassay methods. In exemplary embodiments, such methodsinclude methods for isolating a molecule of interest (such as forexample a specific antibody that is present in a test sample, or aspecific antigen that may be present in the test sample). In order tofacilitate such isolation, the molecule of interest, for example,comprises or is attracted to a purification tag that contacts a tagbinding partner. The association of the purification tag and the tagbinding partner thus may be used to separate the molecule of interestfrom a mixture of molecules. Purification tags can comprise moietieswith the same or similar structures. In certain embodiments, the taggingmoiety of an affinity tag can be associated with a functional tagdirectly by a single bond or via a linkage of stable chemical bonds, inlinear, branched or cyclic arrangements, optionally including single,double, triple bond, aromatic carbon-carbon bonds, as well ascarbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds,carbon-sulfur bonds, phosphorus-oxygen bonds, phosphorus-nitrogen bonds,and any combination thereof. In certain embodiments, the associationbetween the tagging moiety and functional tag comprises ether,thioether, carboxamide, sulfonamide, urea or urethane moieties. Incertain embodiments, the linkage comprises a polyalkylene chain, i.e., alinear or branched arrangement of carbon-carbon bonds. In otherembodiments, the linkage comprises a polyalkylene oxide chain, includinga polyethylene glycol moiety. Examples, of affinity tags include, butare not limited to, biotin, digoxigenin (Dig), dinitrophenol (DNP), zincfingers, fluorinated polymers, and polypeptide sequences such aspolyhistidine motifs.

The affinity tags are, in some embodiments, advantageously used toisolate the molecule of interest by relying on the binding or attractionof the affinity tag and a functional group that is attracted to or bindsthe affinity tag. In some embodiments, solid substrates having anaffinity for the tag in that the solid substrate is derivatized with thetag binding partner. In some embodiments, the binding partner may beimmobilized on an affinity substrate. The term “affinity substrate” canrefer to an immobile matrix or support bound to a binding partner thatis capable of forming a strong and preferably reversible interactionwith the purification tag of a molecule. An affinity substrate caninclude a resin, a bead, a particle, a membrane, a gel. The bindingpartner recognizes or binds to the purification tag specifically.Specific binding partners will depend on the affinity tag, but includecharged moieties and one member of a binding pair such asreceptor-ligand, antibody-antigen, carbohydrate-lectin, andbiotin-streptavidin (or avidin, neutravidin or an anti-biotin antibody).

In specific embodiments, either the C or the N terminus of any or all ofthe antigens used in the immunoassay may be biotinylated or may comprisea biotin binding moiety (e.g., avidin or streptavidin or neutravidin oran anti-biotin) as the affinity tag. These peptides are biotinylated oravidin/streptavidin-conjugated peptides and will serve as captureantigens. Likewise, the antigens may alternatively be labeled with adetection label in which case they will serve as detection antigens. Thedetection and capture antigens may have the same underlying amino acidsequence or alternatively, may have different sequences. In exemplaryembodiments, the capture antigens are biotinylated at either the C orthe N terminus to facilitate binding thereof to solid supports that havethe biotin binding partner (i.e., avidin or streptavidin). For exemplaryproduction purposes, the biotinylated peptides are recombinantlyexpressed in E. coli BL2L(DE3) cells via an IPTG induction system at 25°C. In situ biotinylation at the C-terminal or N-terminal biotinylationis accomplished by co-transformation of the BL21(DE3) cells with the HCVexpression plasmid expressing the desired peptide and a second plasmidcontaining the BirA gene from E. coli (Weiss et al. (1994) ProteinExpression & Purif, 14:751-755; Schatz et al. (1993) Biotechnology,11:1138-1143). Purification of the recombinant proteins is performedusing divalent cation chelators that are shown to preventmetal-catalyzed oxidation and aggregation of the protein. Proteinstability is significantly improved when EDTA or related divalent cationchelator is added to the buffers used during purification and to thefinal storage buffer or buffers used in the immunoassay.

In certain embodiments, besides determining the presence of subjectantibodies in a sample, the assays also determine the presence of one ormore HCV antigens in the sample. In such embodiments, it will bedesirable to use monoclonal anti-HCV antibodies to capture the antigenfrom the test sample and then use further conjugate antibodies to detectthe presence of antigen that has been captured. There are numerouscommercially available antibodies that may be used in this endeavor.Specifically, such antibodies preferably determine the presence of Coreantigen in the test sample. Antibodies directed to Core antigen areknown to those of skill in the art include, for example, those describedin US Patent Publication No. 20120009196 and 20140272931, both of whichare herein incorporated by reference in their entirety.

In specific exemplary embodiments the antibodies used in the combinationimmunoassay are antibodies designed to detect HCV core protein orfragments thereof in a test sample. Such antibodies may detect the DNAbinding domain, the lipid binding domain or the complete Core protein.In some embodiments, the detection antibody used in the immunoassay isdirected to the lipid binding domain of core peptide. In still otherembodiments, the anti-HCV Core antibodies used in the combination assaysmay be for example, C11-3, C11-7, C11-9, and C11-14 (as described inU.S. Pat. No. 6,727,092; Morota, et al, J. Virol. Meth., 2009,157:8-14).

In a specific assay of the present invention, the immunoassay at leastdetects NS3h specific subject antibodies, core antigen, as welldetecting core antibodies in the test sample. In such embodiments, itbecomes desirable, although not essential to ensure that the captureantigen that is designed to capture anti-Core one that preferablycomprise certain deletions or substitution in the known epitope bindingregions for specific monoclonal antibodies such that monoclonalantibodies used for HCV core antigen detection would fail to detectthese modified core antigens but would nonetheless detect complete coreantigen from the test sample. Exemplary anti-core antibodies to be usedas capture antibodies include antibodies AOT3, C11-3, C11-7, C11-9, andC11-14 as described in U.S. Pat. No. 6,727,092 as well as Morota, et al,J. Virol. Meth., 2009, 157:8-14.

In particular embodiments, the antigens and antibodies described hereinare contemplated for use as immunodiagnostic reagents in combinationimmunoassays designed for the detection of multiple HCV components foundin a test sample suspected of having been infected with HCV.Immunodiagnostic reagents (be they antibodies or antigens) will becomprised of the herein-described antigen polypeptides and antibodies(typically in combination) such that they can be used in a combinationimmunoassay designed for the detection of HCV antigens including but notlimited to the NS3 region of HCV, the core antigen of HCV, the NS4region of HCV or combinations thereof as well as anti-HCV antibodiesdirected against one or more of these regions. For purposes of capture,the antigens and/or antibodies of which the immunodiagnostic reagent iscomprised can be coated on a solid support such as for example, amicroparticle, (e.g., magnetic particle), bead, test tube, microtiterplate, cuvette, membrane, scaffolding molecule, film, filter paper, discor chip. In this regard, where the immunodiagnostic reagent comprises acombination of antigens (e.g., directed at different HCV proteins ordifferent fragments of the same HCV protein), the antigens can beco-coated on the same solid support or can be on separate solidsupports. Likewise, where the immunodiagnostic reagent comprises one ormore antibodies that will be used to capture one or more antigens fromthe test sample, such antibodies can be co-coated on the same solidsupport or can be on separate solid supports.

Notably, the immunodiagnostic reagent may be labeled with a detectablelabel or labeled with a specific partner that allows capture ordetection. For example, the labels may be a detectable label, such as afluorophore, radioactive moiety, enzyme, biotin/avidin label,chromophore, chemiluminescent label, or the like. Such labels aredescribed in further detail infra.

Still further the invention contemplates the preparation of HCVdiagnostic kits comprising the immunodiagnostic reagents describedherein and may further include instructions for the use of theimmunodiagnostic reagents in immunoassays for determining the presenceof HCV in a test sample. For example, the kit can comprise instructionsfor assaying the test sample for anti-HCV antibody (e.g., an anti-NS3hantibody in the test sample) by immunoassay. While certain embodimentsemploy chemiluminescent microparticle immunoassay for assaying the testsample, it should be understood that the antigens and antibodies used inthe immunoassays of the present disclosure may be used in any otherimmunoassay formats known to those of skill in the art for determiningthe presence of HCV in a test sample. The instructions can be in paperform or computer-readable form, such as a disk, CD, DVD, or the like.Alternatively or additionally, the kit can comprise a calibrator orcontrol, e.g., purified, and optionally lyophilized, anti-HCV antibodyor antigen, and/or at least one container (e.g., tube, microtiter platesor strips, which can be already coated with one or more of the capturecomponents (antigens and/or antibodies) of the combination immunoassay)for conducting the assay, and/or a buffer, such as an assay buffer or awash buffer, either one of which can be provided as a concentratedsolution, a substrate solution for the detectable label (e.g., anenzymatic label), or a stop solution. Preferably, the kit comprises allcomponents, i.e., reagents, standards, buffers, diluents, etc., whichare necessary to perform the assay. In specific embodiments, it ispreferred that all the components are individually presented in the kitsuch that the immunoassay may be performed as a capture-on-the-fly typeimmunoassay in which the solid support is coated with an agent thatallows binding of the capturing moiety (e.g., a biotinylated antigen ora biotinylated antibody) and the kit further comprises each of theindividual capture and detection antigen pairs and the biotinylatedcapture antibodies in one container and a second container provides thedetection antibody conjugate. The instructions for conducting the assayalso can include instructions for generating a standard curve or areference standard for purposes of quantifying anti-HCV antibody.

Any antibodies, which are provided in the kit or systems herein, such asanti-IgG antibodies and anti-IgM antibodies, can also incorporate adetectable label, such as a fluorophore, radioactive moiety, enzyme,biotin/avidin label, chromophore, chemiluminescent label, or the like,or the kit can include reagents for labeling the antibodies or reagentsfor detecting the antibodies (e.g., detection antibodies) and/or forlabeling the analytes or reagents for detecting the analyte. Theantibodies, calibrators and/or controls can be provided in separatecontainers or pre-dispensed into an appropriate assay format, forexample, into microtiter plates. In one embodiment, there are twocontainers provided. In the first container is provided at least afirst, second and/or third pair of antigens, wherein the first antigenin each pair is a capture antigen from a given HCV protein (e.g., NS3h)that is biotinylated and the second antigen in each pair is a detectionantigen from the same protein as the first antigen but is labeled with adetectable label (e.g., it is acridinylated) as well as one or morebiotinylated antibodies designed for detecting one or more HCV antigensfrom a test sample; and in the second container is provided the antibodythat forms the conjugation partner for detection of the antigen that iscaptured by the biotinylated antibodies from the first container. It iscontemplated that where there are multiple biotinylated antibodies inthe first container, the multiple antibodies that form the conjugationpartners may be present in a single container or individual containersfor each different antigen detecting conjugate antibody.

In certain embodiments, the kit includes quality control components (forexample, sensitivity panels, calibrators, and positive controls).Preparation of quality control reagents is well-known in the art and isdescribed on insert sheets for a variety of immunodiagnostic products.Sensitivity panel members optionally are used to establish assayperformance characteristics, and further optionally are usefulindicators of the integrity of the immunoassay kit reagents, and thestandardization of assays.

The kits may also include other reagents required to conduct adiagnostic assay or facilitate quality control evaluations, such asbuffers, salts, enzymes, enzyme co-factors, substrates, detectionreagents, and the like. Other components, such as buffers and solutionsfor the isolation and/or treatment of a test sample (e.g., pretreatmentreagents), also can be included in the kit. The kit can additionallyinclude one or more other controls. One or more of the components of thekit can be lyophilized, in which case the kit can further comprisereagents suitable for the reconstitution of the lyophilized components.

The various components of the kit may be provided in suitable containersas necessary, e.g., a microtiter plate. The kit can further includecontainers for holding or storing a sample (e.g., a container orcartridge for a sample). Where appropriate, the kit optionally also cancontain reaction vessels, mixing vessels, and other components thatfacilitate the preparation of reagents or the test sample. The kit canalso include one or more instrument for assisting with obtaining a testsample, such as a syringe, pipette, forceps, measured spoon, or thelike.

In certain embodiments, the detectable label is at least one acridiniumcompound. In such embodiments, the kit can comprise at least oneacridinium-9-carboxamide, at least one acridinium-9-carboxylate arylester, or any combination thereof. If the detectable label is at leastone acridinium compound, the kit also can comprise a source of hydrogenperoxide, such as a buffer, solution, and/or at least one basicsolution. It should be understood that in the immunodiagnostic reagentthe antigens for antibody detection may be detectably labeled, and anyantibodies provided in kit for use along with such reagents also may bedetectably labeled.

In certain embodiments, the kit can contain a solid support phase, suchas a magnetic particle, bead, test tube, microtiter plate, cuvette,membrane, scaffolding molecule, film, filter paper, disc or chip.

The present disclosure provides immunoassay methods for determining thepresence, amount or concentration of anti-HCV antibodies or anti-HCVantibodies and HCV antigens in a test sample. Any suitable assay knownin the art can be used in such a method as long as such an assay usesone or more of antigens for detecting HCV antibodies. Examples of suchassays include, but are not limited to, immunoassay, such as sandwichimmunoassay (e.g., monoclonal-polyclonal sandwich immunoassays,including radioisotope detection (radioimmunoassay (RIA)) and enzymedetection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay(ELISA) (e.g., Quantikine ELISA assays, R&D Systems, Minneapolis,Minn.)), competitive inhibition immunoassay (e.g., forward and reverse),fluorescence polarization immunoassay (FPIA), enzyme multipliedimmunoassay technique (EMIT), bioluminescence resonance energy transfer(BRET), and homogeneous chemiluminescent assay, etc.

In particular embodiments, the recombinant antigens (e.g., NS3hantigens) may be used as capture reagents (e.g., by using such antigensin which the amino- or carboxy-terminal of the antigen comprises abiotin tag) or as a detection (conjugate) reagents in which the antigensare either directly or indirectly labeled with acridinium. Indirectlabeling may employ the use of acridinylated BSA covalently coupled tothe free thiol of unpaired cysteine residues within a protein viaSMCC-type linker. To facilitate such indirect labeling certain of theantigens used in the immunoassays of the present disclosure may readilybe further modified to include additional cysteine residues at theC-terminus.

Typically, immunoassays are performed in 1-step or 2-step format. Solidphase reagents for capture of immune complexes formed in solution in the1-step assay include, for example, anti-biotin monoclonal antibody,streptavidin or neutravidin to capture the biotinylated moiety (e.g., abiotinylated antigen for capture of an HCV antibody).

In a SELDI-based immunoassay, a capture reagent that specifically bindsanti-HCV-antibody or an HCV antigen is attached to the surface of a massspectrometry probe, such as a pre-activated protein chip array. Theanti-HCV antibody or the antigen is then specifically captured on thebiochip, and the captured moiety is detected by mass spectrometry.Alternatively, the anti-HCV antibody can be eluted from the capturereagent and detected by traditional MALDI (matrix-assisted laserdesorption/ionization) or by SELDI. A chemiluminescent microparticleimmunoassay, in particular one employing the ARCHITECT® automatedanalyzer (Abbott Laboratories, Abbott Park, Ill.), is an example of aparticular immunoassay in which a combination of multiple antigens(preferably two or more NS3h antigens) may readily be employed. Anagglutination assay, such as a passive hemagglutination assay, also canbe used. In an agglutination assay an antigen-antibody reaction isdetected by agglutination or clumping. In a passive hemagglutinationassay, erythrocytes are coated with the antigen and the coatederythrocytes are used in the agglutination assay.

Methods well-known in the art for collecting, handling and processingurine, blood, serum and plasma, and other body fluids, are used in thepractice of the present disclosure, for instance, when theimmunodiagnostic reagents comprise multiple antigens and/or in ananti-HCV antibody immunoassay kit. The test sample can comprise furthermoieties in addition to the polypeptide of interest, such as antibodies,antigens, haptens, hormones, drugs, enzymes, receptors, proteins,peptides, polypeptides, oligonucleotides or polynucleotides. Forexample, the sample can be a whole blood sample obtained from a subject.It can be necessary or desired that a test sample, particularly wholeblood, be treated prior to immunoassay as described herein, e.g., with apretreatment reagent. Even in cases where pretreatment is not necessary(e.g., most urine samples), pretreatment optionally can be done for mereconvenience (e.g., as part of a regimen on a commercial platform).

The pretreatment reagent can be any reagent appropriate for use with theimmunoassays and kits described herein. The pretreatment optionallycomprises: (a) one or more solvents (e.g., methanol and ethylene glycol)and salt, (b) one or more solvents, salt and detergent, (c) detergent,or (d) detergent and salt. Pretreatment reagents are known in the art,and such pretreatment can be employed, e.g., as used for assays onAbbott TDx, AxSYM®, and ARCHITECT® analyzers (Abbott Laboratories,Abbott Park, Ill.), as described in the literature (see, e.g., Yatscoffet al., Abbott TDx Monoclonal Antibody Assay Evaluated for MeasuringCyclosporine in Whole Blood, Clin. Chem. 36: 1969-1973 (1990), andWallemacq et al., Evaluation of the New AxSYM Cyclosporine Assay:Comparison with TDx Monoclonal Whole Blood and EMIT Cyclosporine Assays,Clin. Chem. 45: 432-435 (1999)), and/or as commercially available.Additionally, pretreatment can be done as described in Abbott's U.S.Pat. No. 5,135,875, European Pat. Pub. No. 0 471 293, U.S. ProvisionalPat. App. 60/878,017, filed Dec. 29, 2006, and U.S. Pat. App. Pub. No.2008/0020401 (incorporated by reference in its entirety for itsteachings regarding pretreatment). The pretreatment reagent can be aheterogeneous agent or a homogeneous agent.

With use of a heterogeneous pretreatment reagent, the pretreatmentreagent precipitates analyte binding protein (e.g., protein that canbind to anti-HCV antibody or an antigen that can bind to an anti-HCVantibody form the present in the sample). Such a pretreatment stepcomprises removing any analyte binding protein by separating from theprecipitated analyte binding protein the supernatant of the mixtureformed by addition of the pretreatment agent to sample. In such anassay, the supernatant of the mixture absent any binding protein is usedin the assay, proceeding directly to the antibody capture step.

With use of a homogeneous pretreatment reagent there is no suchseparation step. The entire mixture of test sample and pretreatmentreagent are contacted with a labeled specific binding partner foranti-HCV antibody (e.g., an antigen) or the labeled specific bindingpartner for the HCV antigen (e.g., an antibody). The pretreatmentreagent employed for such an assay typically is diluted in thepretreated test sample mixture, either before or during capture by thefirst specific binding partner. Despite such dilution, a certain amountof the pretreatment reagent (for example, 5 M methanol and/or 0.6methylene glycol) is still present (or remains) in the test samplemixture during capture.

In a heterogeneous format, after the test sample is obtained from asubject, a first mixture is prepared. The mixture contains the testsample being assessed for anti-HCV antibodies and a first specificcapture binding partner, wherein the first specific capture bindingpartner and any anti-HCV antibodies contained in the test sample form afirst specific capture binding partner-anti-HCV antibody complex. Thefirst specific capture binding partner may be an NS3 antigen. Likewise,in the assays provided herein may also contain a second and thirdspecific capture binding partner and these second and third specificcapture binding partners form second and third specific capture bindingpartner-anti-HCV antibody complexes with anti-HCV antibodies that arepresent in the test sample. Such second, third and fourth antigens maybe one or more of at least one HCV antigen selected from the groupconsisting of core antigen, NS3, NS4, NS5, and portions thereof.

In addition immunoassays, besides includes at least one NS3h captureantigens, may include at least one anti-HCV capture antibody that willform a specific complex with a specific binding partner that is found inthe test sample (i.e., an antigen or HCV protein that is found in thetest sample) so as to form an anti-HCV antibody-third or fourth specificbinding partner complex with the antigen that is present in the testsample. In certain embodiments, this specific binding pair is one thatdetects Core antigen in a test sample, and hence the binding pair is ananti-Core antibody for detection of the antigen (Core) in the testsample.

The order in which the test sample and the various specific bindingpartners are added to form the mixture may vary. In some embodiments,the first, second, third, etc. specific capture binding partners (i.e.,antigens) and any anti-HCV capture antibody are immobilized on a solidphase. In still other embodiments, none of these components areimmobilized but are instead all added at the same time to the testsample to effect capture onto the solid phase. The solid phase used inthe immunoassay can be any solid phase known in the art, such as, butnot limited to, a magnetic particle, a bead, a test tube, a microtiterplate, a cuvette, a membrane, a scaffolding molecule, a film, a filterpaper, a disc and a chip.

After the immunocomplexes are formed between the specific capturebinding partners and their respective anti-HCV antibodies found in thetest sample, and any anti-HCV capture antibodies (e.g., anti-Core) andtheir respective HCV antigens or HCV proteins found in the test sample,any unbound anti-HCV antibody or HCV antigen/protein is removed from thecomplex using any technique known in the art. For example, the unboundanti-HCV antibody or antigen can be removed by washing. Desirably,however, the specific binding partners and any anti-HCV antibodies arepresent in excess of any anti-HCV antibody and antigens, respectivelypresent in the test sample, such that all anti-HCV antibody and antigensthat are present in the test sample become bound by the specific bindingpartner and any anti-HCV capture antibodies respectively.

After any unbound anti-HCV antibody and antigen is removed, detection isachieved by addition of a first specific detection binding partner(e.g., conjugate) to the mixture to form a first specific capturebinding partner-anti-HCV antibody-first specific detection bindingpartner complex. The first specific detection binding partner may be alabeled antigen (e.g., NS3h antigen) or an anti-IgG antibody or ananti-IgM antibody. Moreover, the first specific detection bindingpartner may be labeled with or contain a detectable label as describedabove.

Any suitable detectable label as is known in the art can be used as anyone or more of the detectable labels. For example, the detectable labelcan be a radioactive label (such as 3H, 125I, 35S, 14C, 32P, and 33P),an enzymatic label (such as horseradish peroxidase, alkaline peroxidase,glucose 6-phosphate dehydrogenase, and the like), a chemiluminescentlabel (such as acridinium esters, thioesters, or sulfonamides; luminol,isoluminol, phenanthridinium esters, and the like), a fluorescent label(such as fluorescein (e.g., 5-fluorescein, 6-carboxyfluorescein,3′6-carboxyfluorescein, 5(6)-carboxyfluorescein,6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluoresceinisothiocyanate, and the like)), rhodamine, phycobiliproteins,R-phycoerythrin, quantum dots (e.g., zinc sulfide-capped cadmiumselenide), a thermometric label, or an immuno-polymerase chain reactionlabel. An introduction to labels, labeling procedures and detection oflabels is found in Polak and Van Noorden, Introduction toImmunocytochemistry, 2nd ed., Springer Verlag, N.Y. (1997), and inHaugland, Handbook of Fluorescent Probes and Research Chemicals (1996),which is a combined handbook and catalogue published by MolecularProbes, Inc., Eugene, Oreg. A fluorescent label can be used in FPIA(see, e.g., U.S. Pat. Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093,and 5,352,803, which are hereby incorporated by reference in theirentireties). An acridinium compound can be used as a detectable label ina homogeneous chemiluminescent assay (see, e.g., Adamczyk et al.,Bioorg. Med. Chem. Lett. 16: 1324-1328 (2006); Adamczyk et al., Bioorg.Med. Chem. Lett. 4: 2313-2317 (2004); Adamczyk et al., Biorg. Med. Chem.Lett. 14: 3917-3921 (2004); and Adamczyk et al., Org. Lett. 5: 3779-3782(2003)).

An exemplary acridinium compound is an acridinium-9-carboxamide. Methodsfor preparing acridinium 9-carboxamides are described in Mattingly, J.Biolumin. Chemilumin. 6: 107-114 (1991); Adamczyk et al., J. Org. Chem.63: 5636-5639 (1998); Adamczyk et al., Tetrahedron 55: 10899-10914(1999); Adamczyk et al., Org. Lett. 1: 779-781 (1999); Adamczyk et al.,Bioconjugate Chem. 11: 714-724 (2000); Mattingly et al., In LuminescenceBiotechnology: Instruments and Applications; Dyke, K. V. Ed.; CRC Press:Boca Raton, pp. 77-105 (2002); Adamczyk et al., Org. Lett. 5: 3779-3782(2003); and U.S. Pat. Nos. 5,468,646, 5,543,524 and 5,783,699 (each ofwhich is incorporated herein by reference in its entirety for itsteachings regarding same).

Another exemplary acridinium compound is an acridinium-9-carboxylatearyl ester. An example of an acridinium-9-carboxylate aryl ester offormula II is 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate(available from Cayman Chemical, Ann Arbor, Mich.). Methods forpreparing acridinium 9-carboxylate aryl esters are described in McCapraet al., Photochem. Photobiol. 4: 1111-21 (1965); Razavi et al.,Luminescence 15: 245-249 (2000); Razavi et al., Luminescence 15: 239-244(2000); and U.S. Pat. No. 5,241,070 (each of which is incorporatedherein by reference in its entirety for its teachings regarding same).Such acridinium-9-carboxylate aryl esters are efficient chemiluminescentindicators for hydrogen peroxide produced in the oxidation of an analyteby at least one oxidase in terms of the intensity of the signal and/orthe rapidity of the signal. The course of the chemiluminescent emissionfor the acridinium-9-carboxylate aryl ester is completed rapidly, i.e.,in under 1 second, while the acridinium-9-carboxamide chemiluminescentemission extends over 2 seconds. Acridinium-9-carboxylate aryl ester,however, loses its chemiluminescent properties in the presence ofprotein. Therefore, its use requires the absence of protein duringsignal generation and detection. Methods for separating or removingproteins in the sample are well-known to those skilled in the art andinclude, but are not limited to, ultrafiltration, extraction,precipitation, dialysis, chromatography, and/or digestion (see, e.g.,Wells, High Throughput Bioanalytical Sample Preparation. Methods andAutomation Strategies, Elsevier (2003)). The amount of protein removedor separated from the test sample can be about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, or about 95%. Further details regardingacridinium-9-carboxylate aryl ester and its use are set forth in U.S.patent application Ser. No. 11/697,835, filed Apr. 9, 2007, andpublished on Oct. 9, 2008, as U.S. Pat. App. Pub. No. 2008/0248493.Acridinium-9-carboxylate aryl esters can be dissolved in any suitablesolvent, such as degassed anhydrous N,N-dimethylformamide (DMF) oraqueous sodium cholate.

Chemiluminescent assays can be performed in accordance with the methodsdescribed in Adamczyk et al., Anal. Chim. Acta 579(1): 61-67 (2006).While any suitable assay format can be used, a microplatechemiluminometer (Mithras LB-940, Berthold Technologies U.S.A., LLC, OakRidge, Tenn.) enables the assay of multiple samples of small volumesrapidly. The chemiluminometer can be equipped with multiple reagentinjectors using 96-well black polystyrene microplates (Costar #3792).Each sample can be added into a separate well, followed by thesimultaneous/sequential addition of other reagents as determined by thetype of assay employed. Desirably, the formation of pseudobases inneutral or basic solutions employing an acridinium aryl ester isavoided, such as by acidification. The chemiluminescent response is thenrecorded well-by-well. In this regard, the time for recording thechemiluminescent response will depend, in part, on the delay between theaddition of the reagents and the particular acridinium employed.

Hydrogen peroxide can be generated in situ in the mixture or provided orsupplied to the mixture before, simultaneously with, or after theaddition of an above-described acridinium compound. Hydrogen peroxidecan be generated in situ in a number of ways such as would be apparentto one skilled in the art. Alternatively, a source of hydrogen peroxidecan be simply added to the mixture. For example, the source of thehydrogen peroxide can be one or more buffers or other solutions that areknown to contain hydrogen peroxide. In this regard, a solution ofhydrogen peroxide can simply be added. Upon the simultaneous orsubsequent addition of at least one basic solution to the sample, adetectable signal, namely, a chemiluminescent signal, indicative of thepresence of anti-HCV antibody (where capture is with an antigen) orantigen (where capture is with an antibody) is generated. The basicsolution contains at least one base and has a pH greater than or equalto 10, preferably, greater than or equal to 12. Examples of basicsolutions include, but are not limited to, sodium hydroxide, potassiumhydroxide, calcium hydroxide, ammonium hydroxide, magnesium hydroxide,sodium carbonate, sodium bicarbonate, calcium hydroxide, calciumcarbonate, and calcium bicarbonate. The amount of basic solution addedto the sample depends on the concentration of the basic solution. Basedon the concentration of the basic solution used, one skilled in the artcan easily determine the amount of basic solution to add to the sample.

The chemiluminescent signal that is generated can be detected usingroutine techniques known to those skilled in the art. Based on theintensity of the signal generated, the amount of anti-HCV antibodyand/or antigen in the sample can be quantified. Specifically, the amountof anti-HCV antibody and/or in the sample is proportional to theintensity of the signal generated. The amount of anti-HCV antibodyand/or antigen present can be quantified by comparing the amount oflight generated to a standard curve for anti-HCV antibody and/or antigenor by comparison to a reference standard. The standard curve can begenerated using serial dilutions or solutions of known concentrations ofanti-HCV antibody by mass spectroscopy, gravimetric methods, and othertechniques known in the art.

Anti-HCV antibody and/or antigen immunoassays can be conducted using anysuitable format known in the art. Generally speaking, in certainembodiments, a sample being tested for (for example, suspected ofcontaining) anti-HCV antibodies can be contacted with a capture antigenand at least one detection conjugate peptide or conjugate antibody(which can be a second detection antibody or a third detectionantibody), such as labeled anti-IgG and anti-IgM antibodies, eithersimultaneously or sequentially and in any order. Similarly, the test forpresence of an antigen can be contacted with a captured antibody whichbinds the antigen in the test sample and the bound antigen may then bedetected by a detection antibody.

For example, the test sample can be first contacted with at least twoNS3h capture antigens and then (sequentially) with at least onedetection antibody. Alternatively, the test sample can be firstcontacted with at least one detection antibody and then (sequentially)with at least two NS3h capture antigens. In yet another alternative, thetest sample can be contacted simultaneously with a capture antigen and adetection antibody. In the sandwich assay format, a sample suspected ofcontaining anti-HCV antibodies (or a fragment thereof) is first broughtinto contact with an at least two NS3h capture antigens under conditionsthat allow the formation of a first capture antigen/anti-HCV antibodycomplex and a second antigen/anti-HCV antibody complex. In certainassays, the same is repeated or simultaneously conducted with a second,third or more capture antigens. In a sandwich assay, the antigens)preferably, the at least two NS3h capture antigens are used in molarexcess amounts of the maximum amount of anti-HCV antibodies expected inthe test sample. For example, from about 5 ug to about 1 mg of antigenper mL of buffer (e.g., microparticle coating buffer) can be used.

Competitive inhibition immunoassays, which are often used to measuresmall analytes, comprise sequential and classic formats. In a sequentialcompetitive inhibition immunoassay at least one or at least two NS3hcapture antigens are coated onto a well of a microtiter plate. When thesample containing the antibody/antibodies of interest is added to thewell, the antibody of interest binds to the capture antigens. Afterwashing, a known amount of labeled (e.g., biotin or horseradishperoxidase (HRP)) antibody is added to the well. A substrate for anenzymatic label is necessary to generate a signal. An example of asuitable substrate for HRP is 3,3′,5,5′-tetramethylbenzidine (TMB).After washing, the signal generated by the labeled antibody is measuredand is inversely proportional to the amount of antibody in the sample.In a classic competitive inhibition immunoassay antigen for an antibodyof interest is coated onto a well of a microtiter plate. However, unlikethe sequential competitive inhibition immunoassay, the sample containingthe antibody of interest (i.e., an anti-HCV antibody) and the labeledantibody are added to the well at the same. Any antibody in the samplecompetes with labeled antibody for binding to the capture antigen. Afterwashing, the signal generated by the labeled analyte is measured and isinversely proportional to the amount of analyte in the sample.

In other embodiments, prior to contacting the test sample with the atleast one or at least two NS3h capture antigens, the capture antigenscan be bound to a solid support, which facilitates the separation of thefirst and second antigen/anti-HCV antibody complexes from the testsample. The substrate to which the capture antigens are bound can be anysuitable solid support or solid phase that facilitates separation of thecapture antigen-anti-HCV antibody complexes from the sample. Examplesinclude a well of a plate, such as a microtiter plate, a test tube, aporous gel (e.g., silica gel, agarose, dextran, or gelatin), a polymericfilm (e.g., polyacrylamide), beads (e.g., polystyrene beads or magneticbeads), a strip of a filter/membrane (e.g., nitrocellulose or nylon),microparticles (e.g., latex particles, magnetizable microparticles(e.g., microparticles having ferric oxide or chromium oxide cores andhomo- or hetero-polymeric coats and radii of about 1-10 microns). Thesubstrate can comprise a suitable porous material with a suitablesurface affinity to bind antigens and sufficient porosity to allowaccess by detection antibodies. A microporous material is generallypreferred, although a gelatinous material in a hydrated state can beused. Such porous substrates are preferably in the form of sheets havinga thickness of about 0.01 to about 0.5 mm, preferably about 0.1 mm.While the pore size may vary quite a bit, preferably the pore size isfrom about 0.025 to about 15 microns, more preferably from about 0.15 toabout 15 microns. The surface of such substrates can be activated bychemical processes that cause covalent linkage of an antibody to thesubstrate. Irreversible binding, generally by adsorption throughhydrophobic forces, of the antigen to the substrate results;alternatively, a chemical coupling agent or other means can be used tobind covalently the antigen to the substrate, provided that such bindingdoes not interfere with the ability of the antigen to bind to anti-HCVantibodies.

In other embodiments, the anti-HCV antibodies from the test sample canbe bound with microparticles, which have been previously coated withantigen. If desired, one or more capture reagents, such as one or moreor two or more NS3h antigens as described herein, each of which can bebound by an anti-HCV antibody, can be attached to solid phases indifferent physical or addressable locations (e.g., such as in a biochipconfiguration (see, e.g., U.S. Pat. No. 6,225,047, Int'l Pat. App. Pub.No. WO 99/51773; U.S. Pat. No. 6,329,209; Int'l Pat. App. Pub. No. WO00/56934, and U.S. Pat. No. 5,242,828). If the capture reagents areattached to a mass spectrometry probe as the solid support, the amountof anti-HCV antibodies bound to the probe can be detected by laserdesorption ionization mass spectrometry. Alternatively, a single columncan be packed with different beads, which are derivatized with the oneor more capture reagents, thereby capturing the anti-HCV antibody in asingle place (see, antibody derivatized, bead-based technologies, e.g.,the xMAP technology of Luminex (Austin, Tex.)).

In certain embodiments, after the test sample being assayed for anti-HCVantibodies is brought into contact with at least one or at least twoNS3h antigens, the mixture is incubated in order to allow for theformation of a first and second antigen-anti-HCV antibody complexes. Theincubation can be carried out, for example, at a pH of from about 4.5 toabout 10.0, at a temperature of from about 2° C. to about 45° C., andfor a period from at least about one (1) minute to about eighteen (18)hours, or from about 1 to about 24 minutes, or for about 4 to about 18minutes.

In certain embodiments, at least one detection antibody is employed andcontains a detectable label. The detectable label can be bound to the atleast one detection antibody prior to, simultaneously with, or after theformation of the (first or multiple) capture antigen/anti-HCVantibody/(second or multiple) detection antibody complex. Any detectablelabel known in the art can be used (see discussion above, includingPolak and Van Noorden (1997) and Haugland (1996)). The detectable labelcan be bound to the antibodies (or antigens which may comprisedetectable labels) either directly or through a coupling agent. Anexample of a coupling agent that can be used is EDAC(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, hydrochloride), whichis commercially available from Sigma-Aldrich, St. Louis, Mo. Othercoupling agents that can be used are known in the art. Methods forbinding a detectable label to an antibody are known in the art.Additionally, many detectable labels can be purchased or synthesizedthat already contain end groups that facilitate the coupling of thedetectable label to the antibody, such as CPSP-Acridinium Ester (i.e.,9[N-tosyl-N-(3-carboxypropyl)]-10-(3-sulfopropyl)acridinium carboxamide)or SPSP-Acridinium Ester (i.e.,N10-(3-sulfopropyl)-N-(3-sulfopropyl)-acridinium-9-carboxamide).

The first and second (or more) capture antigen/anti-HCV antibody/labeledconjugate antigen complex can be, but does not have to be, separatedfrom the remainder of the test sample prior to quantification of thelabel. For example, if the capture antigens are bound to a solidsupport, such as a well or a bead, separation can be accomplished byremoving the fluid (of the test sample) from contact with the solidsupport. Alternatively, if the capture antigens are bound to a solidsupport, it can be simultaneously contacted with the anti-HCVantibody-containing sample and a labeled conjugate peptide to form acomplex, followed by removal of the fluid (test sample) from contactwith the solid support. If the capture antigens are not bound to a solidsupport, then complex does not have to be removed from the test samplefor quantification of the amount of the label.

After formation of the detection complexes (e.g., captureantigen/subject antibody/labeled conjugate antigen), the amount of labelin the complex may be quantified using techniques known in the art. Forexample, if an enzymatic label is used, the labeled complex is reactedwith a substrate for the label that gives a quantifiable reaction suchas the development of color. If the label is a radioactive label, thelabel is quantified using a scintillation counter. If the label is afluorescent label, the label is quantified by stimulating the label witha light of one color (which is known as the “excitation wavelength”) anddetecting another color (which is known as the “emission wavelength”)that is emitted by the label in response to the stimulation. If thelabel is a chemiluminescent label, the label is quantified by detectingthe light emitted either visually or by using luminometers, x-ray film,high speed photographic film, a CCD camera, etc. Once the amount of thelabel in the complex has been quantified, the concentration of anti-HCVantibody or antigen in the test sample is determined, for example, byuse of a standard curve that has been generated using serial dilutionsof anti-HCV antibody or antigens of known concentration. Other thanusing serial dilutions of anti-HCV antibodies or HCV antigens, thestandard curve can be generated gravimetrically, by mass spectroscopyand by other techniques known in the art.

In a chemiluminescent microparticle assay employing the ARCHITECT®analyzer, the conjugate diluent pH should generally be about 6.0+/−0.2,the microparticle coating buffer should be maintained at roomtemperature (i.e., at about 17 to about 27° C.), the microparticlecoating buffer pH should be about 6.5+/−0.2, and the microparticlediluent pH should be about 6.5+/−0.2. Solids preferably are less thanabout 0.2%, such as less than about 0.15%, less than about 0.14%, lessthan about 0.13%, less than about 0.12%, or less than about 0.11%, suchas about 0.10%.

Commercially available anti-HCV antibodies as well as anti-IgG andanti-IgM antibodies can be used in the methods of assay and kitsthereof. Commercially available antibodies include those available fromAbnova (Walnut, Calif., and Taiwan) and GenWay Biotech, Inc. (San Diego,Calif.). See, also, European Pat. App. EP2099825 A2 regarding thepreparation of anti-HCV antibodies. Any suitable control composition canbe used in the anti-HCV antibody and HCV antigen immunoassays. Thecontrol composition generally comprises anti-HCV antibodies and knownantigens and any desirable additives.

Generally, a predetermined level can be employed as a benchmark againstwhich to assess results obtained upon assaying a test sample foranti-HCV antibodies. Generally, in making such a comparison, thepredetermined level is obtained by running a particular assay asufficient number of times and under appropriate conditions such that alinkage or association of analyte presence, amount or concentration witha particular stage or endpoint of a disease, disorder or condition(e.g., preeclampsia or cardiovascular disease) or with particularindicia can be made. Typically, the predetermined level is obtained withassays of reference subjects (or populations of subjects).

In particular, with respect to a predetermined level as employed formonitoring disease progression and/or treatment, the amount orconcentration of anti-HCV antibodies may be “unchanged,” “favorable” (or“favorably altered”), or “unfavorable” (or “unfavorably altered”).“Elevated” or “increased” refers to an amount or a concentration in atest sample that is higher than a typical or normal level or range(e.g., predetermined level), or is higher than another reference levelor range (e.g., earlier or baseline sample). The term “lowered” or“reduced” refers to an amount or a concentration in a test sample thatis lower than a typical or normal level or range (e.g., predeterminedlevel), or is lower than another reference level or range (e.g., earlieror baseline sample). The term “altered” refers to an amount or aconcentration in a sample that is altered (increased or decreased) overa typical or normal level or range (e.g., predetermined level), or overanother reference level or range (e.g., earlier or baseline sample).

The typical or normal level or range for anti-HCV antibodies or HCVantigens is defined in accordance with standard practice. Because thelevels of anti-HCV antibodies and/or HCV antigens in some instances willbe very low, a so-called altered level or alteration can be consideredto have occurred when there is any net change as compared to the typicalor normal level or range, or reference level or range, that cannot beexplained by experimental error or sample variation. Thus, the levelmeasured in a particular sample will be compared with the level or rangeof levels determined in similar samples from a so-called normal subject.In this context, a “normal subject” is an individual with no detectablehepatitis, for example, and a “normal” (sometimes termed “control”)patient or population is/are one(s) that exhibit(s) no detectablehepatitis, for example. Furthermore, given that anti-HCV antibodies andHCV antigens are not routinely found at a high level in the majority ofthe human population, a “normal subject” can be considered an individualwith no substantial detectable increased or elevated amount orconcentration of anti-HCV antibodies or HCV antigens, and a “normal”(sometimes termed “control”) patient or population is/are one(s) thatexhibit(s) no substantial detectable increased or elevated amount orconcentration of anti-HCV antibodies. An “apparently normal subject” isone in which anti-HCV antibodies or HCV antigen has not been or is beingassessed. The level of an analyte is said to be “elevated” when theanalyte is normally undetectable (e.g., the normal level is zero, orwithin a range of from about 25 to about 75 percentiles of normalpopulations), but is detected in a test sample, as well as when theanalyte is present in the test sample at a higher than normal level.Thus, inter alia, the disclosure provides a method of screening for asubject having, or at risk of having, hepatitis, for example, as definedherein.

Accordingly, the methods described herein also can be used to determinewhether or not a subject has or is at risk of developing hepatitis.Specifically, such a method can comprise the steps of: (a) determiningthe concentration or amount in a test sample from a subject of anti-HCVantibodies (e.g., using the methods described herein); and (b) comparingthe concentration or amount of anti-HCV antibodies determined in step(a) with a predetermined level, wherein, if the concentration or amountof anti-HCV antibodies and/or HCV antigens determined in step (a) isfavorable with respect to a predetermined level, then the subject isdetermined not to have or be at risk for hepatitis. However, if theconcentration or amount of anti-HCV antibodies in step (a) isunfavorable with respect to the predetermined level, then the subject isdetermined to have or be at risk for hepatitis.

In certain embodiments, the methods described herein can be used tomonitor treatment in a subject receiving treatment with one or morepharmaceutical compositions. Specifically, such methods involveproviding a first test sample from a subject before the subject has beenadministered one or more pharmaceutical compositions. Next, theconcentration or amount in a first test sample from a subject ofanti-HCV antibodies (and optionally HCV antigens) is determined (e.g.,using the at least one or at least two NS3h capture peptide methodsdescribed herein). After the concentration or amount of anti-HCVantibodies is determined, optionally the concentration or amount ofanti-HCV antibodies is then compared with a predetermined level. If theconcentration or amount of anti-HCV antibodies as determined in thefirst test sample is lower than the predetermined level, then thesubject is not treated with one or more pharmaceutical compositions.However, if the concentration or amount of anti-HCV antibodies asdetermined in the first test sample is higher than the predeterminedlevel, then the subject is treated with one or more pharmaceuticalcompositions for a period of time. The period of time that the subjectis treated with the one or more pharmaceutical compositions can bedetermined by one skilled in the art (for example, the period of timecan be from about seven (7) days to about two years, preferably fromabout fourteen (14) days to about one (1) year).

During the course of treatment with the one or more pharmaceuticalcompositions, second and subsequent test samples are then obtained fromthe subject. The number of test samples and the time in which said testsamples are obtained from the subject are not critical. For example, asecond test sample could be obtained seven (7) days after the subject isfirst administered the one or more pharmaceutical compositions, a thirdtest sample could be obtained two (2) weeks after the subject is firstadministered the one or more pharmaceutical compositions, a fourth testsample could be obtained three (3) weeks after the subject is firstadministered the one or more pharmaceutical compositions, a fifth testsample could be obtained four (4) weeks after the subject is firstadministered the one or more pharmaceutical compositions, etc.

After each second or subsequent test sample is obtained from thesubject, the concentration or amount of anti-HCV antibodies (andoptionally HCV antigens) is determined in the second or subsequent testsample is determined. The concentration or amount of anti-HCV antibodiesand/or HCV antigens as determined in each of the second and subsequenttest samples is then compared with the concentration or amount ofanti-HCV antibodies and/or HCV antigens as determined in the first testsample (e.g., the test sample that was originally optionally compared tothe predetermined level). If the concentration or amount of anti-HCVantibodies and/or HCV antigens as determined in step (c) is favorablewhen compared to the concentration or amount of anti-HCV antibodiesand/or HCV antigens as determined in step (a), then the disease in thesubject is determined to have discontinued, regressed or improved, andthe subject should continue to be administered the one or pharmaceuticalcompositions of step (b). However, if the concentration or amountdetermined in step (c) is unchanged or is unfavorable when compared tothe concentration or amount of anti-HCV antibodies and/or HCV antigensas determined in step (a), then the disease in the subject is determinedto have continued, progressed or worsened, and the subject should betreated with a higher concentration of the one or more pharmaceuticalcompositions administered to the subject in step (b) or the subjectshould be treated with one or more pharmaceutical compositions that aredifferent from the one or more pharmaceutical compositions administeredto the subject in step (b). Specifically, the subject can be treatedwith one or more pharmaceutical compositions that are different from theone or more pharmaceutical compositions that the subject had previouslyreceived to decrease or lower said subject's anti-HCV antibodies and/orHCV antigens level.

Generally, for assays in which repeat testing may be done (e.g.,monitoring disease progression and/or response to treatment), a secondor subsequent test sample is obtained at a period in time after thefirst test sample has been obtained from the subject. Specifically, asecond test sample from the subject can be obtained minutes, hours,days, weeks or years after the first test sample has been obtained fromthe subject. For example, the second test sample can be obtained fromthe subject at a time period of about 1 minute, about 5 minutes, about10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours,about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours,about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours,about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days,about 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4 weeks,about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks,about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks,about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks,about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks,about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks,about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2years, about 2.5 years, about 3.0 years, about 3.5 years, about 4.0years, about 4.5 years, about 5.0 years, about 5.5. years, about 6.0years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0years, about 8.5 years, about 9.0 years, about 9.5 years or about 10.0years after the first test sample from the subject is obtained. Whenused to monitor disease progression, the above assay can be used tomonitor the progression of disease in subjects suffering from acuteconditions. Acute conditions, also known as critical care conditions,refer to acute, life-threatening diseases or other critical medicalconditions involving, for example, the cardiovascular system orexcretory system. Typically, critical care conditions refer to thoseconditions requiring acute medical intervention in a hospital-basedsetting (including, but not limited to, the emergency room, intensivecare unit, trauma center, or other emergent care setting) oradministration by a paramedic or other field-based medical personnel.For critical care conditions, repeat monitoring is generally done withina shorter time frame, namely, minutes, hours or days (e.g., about 1minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3hours, about 4 hours, 4 about 5 hours, about 6 hours, about 7 hours,about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours,about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21hours, about 22 hours, about 23 hours, about 24 hours, about 2 days,about 3 days, about 4 days, about 5 days, about 6 days or about 7 days),and the initial assay likewise is generally done within a shortertimeframe, e.g., about minutes, hours or days of the onset of thedisease or condition.

The assays also can be used to monitor the progression of disease insubjects suffering from chronic or non-acute conditions. Non-criticalcare or, non-acute conditions, refers to conditions other than acute,life-threatening disease or other critical medical conditions involving,for example, the cardiovascular system and/or excretory system.Typically, non-acute conditions include those of longer-term or chronicduration. For non-acute conditions, repeat monitoring generally is donewith a longer timeframe, e.g., hours, days, weeks, months or years(e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours,about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours,about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5days, about 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks,about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks,about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks,about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks,about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks,about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2years, about 2.5 years, about 3.0 years, about 3.5 years, about 4.0years, about 4.5 years, about 5.0 years, about 5.5. years, about 6.0years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0years, about 8.5 years, about 9.0 years, about 9.5 years or about 10.0years), and the initial assay likewise generally is done within a longertime frame, e.g., about hours, days, months or years of the onset of thedisease or condition.

Furthermore, the assays described herein can be performed using a firsttest sample obtained from a subject where the first test sample isobtained from one source, such as urine, serum or plasma. Optionally theabove assays can then be repeated using a second test sample obtainedfrom the subject where the second test sample is obtained from anothersource. For example, if the first test sample was obtained from urine,the second test sample can be obtained from serum or plasma. The resultsobtained from the assays using the first test sample and the second testsample can be compared. The comparison can be used to assess the statusof a disease or condition in the subject.

Moreover, the present disclosure also relates to methods of determiningwhether a subject predisposed to or suffering from hepatitis willbenefit from treatment. In particular, the disclosure relates to HCVcompanion diagnostic methods and products. Thus, the method of“monitoring the treatment of disease in a subject” as described hereinfurther optimally also can encompass selecting or identifying candidatesfor therapy. Thus, in particular embodiments, the disclosure alsoprovides a method of determining whether a subject having, or at riskfor, hepatitis is a candidate for therapy. Generally, the subject is onewho has experienced some symptom of hepatitis or who has actually beendiagnosed as having, or being at risk for, hepatitis and/or whodemonstrates an unfavorable concentration or amount of anti-HCVantibodies or a fragment thereof and/or HCV antigens, as describedherein.

The kits and systems (or components thereof), as well as the method ofdetermining the concentration of anti-HCV antibodies and/or HCV antigensin a test sample by an immunoassay as described herein, can be adaptedfor use in a variety of automated and semi-automated systems (includingthose wherein the solid phase comprises a microparticle), as described,e.g., in U.S. Pat. Nos. 5,089,424 and 5,006,309, and as commerciallymarketed, e.g., by Abbott Laboratories (Abbott Park, Ill.) asARCHITECT®. Some of the differences between an automated orsemi-automated system as compared to a non-automated system (e.g.,ELISA) include the substrate to which the first specific binding partner(e.g., antigen) is attached (which can impact sandwich formation andanalyte reactivity), and the length and timing of the capture, detectionand/or any optional wash steps. Whereas a non-automated format such asan ELISA may require a relatively longer incubation time with sample andcapture reagent (e.g., about 2 hours), an automated or semi-automatedformat (e.g., ARCHITECT®, Abbott Laboratories) may have a relativelyshorter incubation time (e.g., approximately 18 minutes for ARCHITECT®).Similarly, whereas a non-automated format such as an ELISA may incubatea detection antibody such as the conjugate reagent for a relativelylonger incubation time (e.g., about 2 hours), an automated orsemi-automated format (e.g., ARCHITECT®) may have a relatively shorterincubation time (e.g., approximately 4 minutes for the ARCHITECT®).Other platforms available from Abbott Laboratories include, but are notlimited to, AxSYM®, IMx® (see, e.g., U.S. Pat. No. 5,294,404, which ishereby incorporated by reference in its entirety), PRISM®, EIA (bead),and Quantum™ II, as well as other platforms. Additionally, the assays,kits and kit components can be employed in other formats, for example,on electrochemical or other hand-held or point-of-care assay systems.The present disclosure is, for example, applicable to the commercialAbbott Point of Care (i-STAT®, Abbott Laboratories) electrochemicalimmunoassay system that performs sandwich immunoassays Immunosensors andtheir methods of manufacture and operation in single-use test devicesare described, for example in, U.S. Pat. No. 5,063,081, U.S. Pat. App.Pub. No. 2003/0170881, U.S. Pat. App. Pub. No. 2004/0018577, U.S. Pat.App. Pub. No. 2005/0054078, and U.S. Pat. App. Pub. No. 2006/0160164,which are incorporated in their entireties by reference for theirteachings regarding same.

EXAMPLES Example 1 Single vs. Multiple NS3 Peptide HCV Assays forVarying HCV Antibody Titer Detection

This Examples describes a comparison of varying HCV antibody titerdetection between HCV assays that employ a single NS3 peptide (withvarious sizes and number of domains), and a combination HCV assay thatemploys both a full-length NS3 helicase peptide and a domain 1 only NS3peptide. The results of this work are shown in Table 1 below.

TABLE 1 ARCHITECT ARCHITECT ARCHITECT Single Marker Assays HCV Ag/AbCombo Anti-HCV NS3 Ab NS3 Ab NS3 Ab (NS3h + NS3h-D1 + (LN 6C37) Core Ag(NS3h) (9NB49) (NS3h-D1) Core Ab Core Ab + Core Ag) Sample ID S/CO S/COS/CO S/CO S/CO S/CO S/CO 11742342  3.99 0.06  2.79  0.06  0.07 0.18 2.28 11742158  9.57 0.04 19.36  0.11  0.11 0.24 16.34 11742354 15.060.06  0.72 11.40 73.48 0.13 62.75

In the 2nd column in Table 1 above, the ARCHITECT Anti-HCV assay wasconducted as follows. In the first step, 10 uL human sample, 90 uL ofassay specific diluent and 50 uL of microparticles coated with HCV NS3,core and NS4 antigens are added to a reaction vessel, vortexed, andincubated for 18 min. Following this incubation, the microparticles aresequestered at the side of the reaction vessel using a magnet while thereaction supernatant is removed. The microparticles are subsequentlywashed with water/detergent solution. During this first step HCVantigen-antibody complexes are formed and captured on themicroparticles. In the second step, immediately following washing, 50 uLof reagent containing acridinylated anti-human IgG and IgM antibodiesare added to the reaction vessel, vortexed and allowed to incubate for 4minutes. During this step the acridinim:anti-human IgG and IgMconjugates further bind to the HCV antigen-antibody complexes formed inthe 1st step. Following incubation, the microparticles are sequesteredat the side of the reaction vessel using a magnet and the reactionsupernatant removed. The microparticles are subsequently washed withwater/detergent solution. Washed particles are suspended in abasic-hydrogen peroxide containing solution to activate the acridiniumwith simultaneous measurement of light output (in relative light unitsor RLU), which is proportional to the amount of conjugate bound onto themicroparticles. The results are reported in Table 1 above, which showsprogressively higher S/COs going from the top of the table down,corresponding to higher titers of antibody in the patient sample.

In the 3rd column in Table 2 above, the ARCHITECT Single Marker Core Agassay was conducted as follows. In the first step, 110 uL human sample,25 uL of assay specific diluent, 50 uL of reagent containingstreptavidin coated microparticles, and 25 uL of reagent containingbiotinylated anti-Core MAb (C11-7) are added to a reaction vessel,vortexed, and incubated for 18 min. During this first step,biotin:anti-core MAb:core Ag complexes are formed and captured on themicroparticles. Following this incubation, the microparticles aresequestered at the side of the reaction vessel using a magnet while thereaction supernatant is removed. The microparticles are subsequentlywashed with water/detergent solution. In the second step, immediatelyfollowing washing, 50 uL of reagent containing acridinylated anti-coreMAbs (C11-9 and C11-14) is added to the reaction vessel, vortexed andallowed to incubate for 4 minutes. During this step theacridinim:anti-core MAb conjugate further binds to the biotin:anti-coreMAb:core Ag complex formed in the 1st step. Following incubation, themicroparticles are sequestered at the side of the reaction vessel usinga magnet and the reaction supernatant removed. The microparticles aresubsequently washed with water/detergent solution. Washed particles aresuspended in a basic-hydrogen peroxide containing solution to activatethe acridinium with simultaneous measurement of light output (inrelative light units or RLU), which is proportional to the amount ofconjugate bound onto the microparticles. The results are reported inTable 1 above, which shows there are no detectable levels of core Ag inthese 3 samples.

In columns 4-7 in Table 1 above, the ARCHITECT Single Marker antibodyassays were conducted as follows. In the first step, 110 uL humansample, 25 uL of reagent containing acridinylated HCV antigen (eitherNS3h-D1 (1192-1356), NS3h-DelN15 (1205-1658), 9NB49 (1192-1457) or corepeptide (15-68, with a deletion at 34 and 48)), 50 uL of reagentcontaining streptavidin coated microparticles, and 25 uL of reagentcontaining biotinylated HCV antigens (either NS3h-D1 (1192-1356),NS3h-DelN15 (1205-1658), 9NB49 (1192-1457) or core peptide (15-68, witha deletion at 34 and 48)), are added to a reaction vessel, vortexed, andincubated for 18 min. During this first step biotin:antigen-HCVantibody-acridinium:antigen complexes are formed and captured on themicroparticles. Following this incubation, the microparticles aresequestered at the side of the reaction vessel using a magnet while thereaction supernatant is removed. The microparticles are subsequentlywashed with water/detergent solution. In the second step, immediatelyfollowing washing, 50 uL of reagent containing a wash solution is addedto the reaction vessel, vortexed and allowed to incubate for 4 minutes.Following incubation, the microparticles are sequestered at the side ofthe reaction vessel using a magnet and the reaction supernatant removed.The microparticles are subsequently washed with water/detergentsolution. Washed particles are suspended in a basic-hydrogen peroxidecontaining solution to activate the acridinium with simultaneousmeasurement of light output (in relative light units or RLU), which isproportional to the amount of conjugate bound onto the microparticles.The results are reported in Table 1 above, which shows the assayutilizing the NS3h-DelN15 (1205-1658) protein can detect the first 2samples of lowest antibody titer while the assays using the NS3h-D1,9NB49 and core peptide cannot. The last sample, of highest antibodytiter, cannot be detected by the assay using the NS3h-DelN15 protein orthe core peptide but can be detected by the assays using the 9NB49 andNS3h-D1 proteins. Furthermore the NS3h-D1 protein shows greaterreactivity for this last sample than the 9NB49 protein.

In the last column in Table 1 above, the HCV Ag/Ab Combo assay wasconducted. Importantly, this assay uses two different types of NS3antigens, including one that contains all three of the helicase domains(e.g., for quantitatively or qualitatively (with a certain cut off)detecting low titer antibody samples), and one that contains only domain1 of the helicase (e.g., for quantitatively or qualitatively detectinghigh titer antibody samples). In the first step, 110 uL human sample, 25uL of reagent containing acridinylated HCV NS3 antigens (both NS3h-D1(1192-1356) and NS3h-DelN15 (1205-1658)) and a core antigen (15-68, witha deletion at 34 and 48), 50 uL of reagent containing streptavidincoated microparticles, and 25 uL of reagent containing biotinylatedanti-Core MAb (C11-7), biotinylated HCV NS3 antigens (both NS3h-D1(1192-1356) and NS3h-DelN15 (1205-1658)), and a biotinylated coreantigen (15-68, with a deletion at 34 and 48), are added to a reactionvessel, vortexed, and incubated for 18 min. During this first stepbiotin:antigen-HCV antibody-acridinium:antigen complexes as well andbiotin:anti-HCV MAb-HCV core Ag complexes are formed and captured on themicroparticles. Following this incubation, the microparticles aresequestered at the side of the reaction vessel using a magnet while thereaction supernatant is removed. The microparticles are subsequentlywashed with water/detergent solution. In the second step, immediatelyfollowing washing, 50 uL of reagent containing acridinylated anti-coreMAbs (C11-9 and C11-14) is added to the reaction vessel, vortexed andallowed to incubate for 4 minutes. During this step theacridinim:anti-HCV MAb conjugate further binds to the biotin:antigen-HCVantibody-acridinium:antigen complex formed in the 1st step. Followingincubation, the microparticles are sequestered at the side of thereaction vessel using a magnet and the reaction supernatant removed. Themicroparticles are subsequently washed with water/detergent solution.Washed particles are suspended in a basic-hydrogen peroxide containingsolution to activate the acridinium with simultaneous measurement oflight output (in relative light units or RLU), which is proportional tothe amount of conjugate bound onto the microparticles. The results arereported in Table 1 above, which shows that when the NS3h-DelN15 andNS3h-D1 proteins are combined into a combo assay all 3 samples aredetected allowing for a larger range of sample antibody concentrationsas compared to using a single NS3 protein alone.

In further work, a serial dilution series of 6 high titer HCV Abpositive samples were tested across two different 1-step anti-HCV NS3assays that utilized 2 NS3 proteins. The NS3h assay, shown in table 2below, utilized the NS3h-DelN15 (1205-1658) protein which has all threeNS3 helicase domains. The NS3h-D1 assay, shown in table 2 below, usedthe NS3h-D1 (1192-1356) which encompasses only domain 1 of the NS3helicase. The results of this testing are shown in Table 2 below.

TABLE 2 HCV Positive Sample Sample ID Sample ID Sample ID Sample IDSample ID Sample ID Sample 11742271 11742348 11742256 11742200 1174235311742193 Dilution NS3h- NS3h- NS3h- NS3h- NS3h- NS3h- Factor (1:x) inNS3h D1 NS3h D1 NS3h D1 NS3h D1 NS3h D1 NS3h D1 Decreasing Ab HCVNegative Assay Assay Assay Assay Assay Assay Assay Assay Assay AssayAssay Assay Concentrations Plasma S/CO S/CO S/CO S/CO S/CO S/CO S/COS/CO S/CO S/CO S/CO S/CO ↓ 1 0.07 36.28 0.12 42.00 0.14 45.63 0.06 28.230.06 21.10 0.08 27.33 10 1.63 67.62 2.33 93.44 5.00 80.75 0.70 71.560.84 70.96 0.74 49.45 100 61.92 37.90 30.20 63.75 98.57 22.91 21.5078.88 29.82 86.80 19.18 49.31 1000 128.06 3.52 125.89 6.20 85.23 1.87112.93 9.65 134.69 12.20 124.80 6.96 10000 30.07 0.29 47.76 0.44 13.460.15 29.29 0.95 43.59 1.05 45.87 0.59 100000 3.16 0.04 5.66 0.05 1.350.03 2.56 0.11 4.34 0.11 5.04 0.07 1000000 2.99 0.02 0.69 0.02 0.15 0.020.26 0.02 0.45 0.03 0.51 0.02

As shown in Table 2, the 1-step assay composed of the single NS3 domain(D1) detected the higher concentrations of Ab but not the lowerconcentrations. The opposite is true for the assay composed of the fulllength NS3h protein that detects the lower antibody concentrationsrather than the high antibody titers.

All publications and patents mentioned in the present application areherein incorporated by reference. Various modification and variation ofthe described methods and compositions of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention that are obvious to those skilledin the relevant fields are intended to be within the scope of thefollowing claims.

We claim:
 1. A system comprising: a) a first NS3h capture peptide thatis no more than 200 amino acids in length and comprises the amino acidsequence of SEQ ID NO: 2 or SEQ ID NO: 3, b) a second NS3h capturepeptide that is no more than 500 amino acids in length and comprises theamino acid sequence of SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9,wherein each of the first and second NS3h capture peptides is able tobind to at least one subject antibody in a biological sample to formcapture complexes, c) a first conjugate peptide that is no more than 200amino acids in length and comprises the amino acid sequence of SEQ IDNO: 2 or SEQ ID NO: 3, and d) a second conjugate peptide that is no morethan 500 amino acids in length and comprises the amino acid sequence ofSEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, wherein each of the firstand second conjugate peptides (1) is able to bind to the at least onesubject antibody in the capture complexes and (2) comprises a detectablelabel, and d) instructions for determining the presence of HCV in thebiological sample.
 2. The system of claim 1, wherein the at least onesubject antibody is not present in the biological sample in purifiedform.
 3. The system of claim 1, wherein the first NS3h capture peptidecomprises the amino acid sequence of SEQ ID NO:
 2. 4. The system ofclaim 1, wherein the first NS3h capture peptide comprises the amino acidsequence of SEQ ID NO:
 3. 5. The system of claim 1, wherein the secondNS3h capture peptide comprises the amino acid sequence of SEQ ID NO: 5.6. The system of claim 1, wherein the second NS3h capture peptidecomprises the amino acid sequence of SEQ ID NO:
 7. 7. The system ofclaim 1, wherein the second NS3h capture peptide comprises the aminoacid sequence of SEQ ID NO:
 9. 8. The system of claim 1, wherein thefirst conjugate peptide comprises the amino acid sequence of SEQ ID NO:2.
 9. The system of claim 1, wherein the first conjugate peptidecomprises the amino acid sequence of SEQ ID NO:
 3. 10. The system ofclaim 1, wherein the second conjugate peptide comprises the amino acidsequence of SEQ ID NO: 5 and a detectable label.
 11. The system of claim1, wherein the second conjugate peptide comprises the amino acidsequence of SEQ ID NO: 7 and a detectable label.
 12. The system of claim1, wherein the second conjugate peptide comprises the amino acidsequence of SEQ ID NO: 9 and a detectable label.
 13. A method ofdetecting hepatitis C virus (HCV) infection in a subject comprising: a)contacting a biological sample with the system of claim 1 to generate amixed biological sample that comprises the biological sample, the firstand second NS3h capture peptides, and the first and second conjugatepeptides, wherein the biological sample is suspected of containingsubject antibodies, and wherein the subject antibodies are not inpurified form in the biological sample, b) incubating the mixedbiological sample under conditions such that: (i) the first NS3h capturepeptide specifically binds to at least one of the subject antibodies toform a first capture complex and the first conjugate peptide binds tothe subject antibody in the first capture complex to form a firstdetectable complex, and (ii) the second NS3h capture peptidespecifically binds to at least one of the subject antibodies to form asecond capture complex and the second conjugate peptide binds to thesubject antibody in the second capture complex to form a seconddetectable complex; c) washing the mixed biological sample to generate awashed sample; and d) detecting the presence of the first and/or seconddetectable complexes, thereby detecting the presence of past or presentHCV infection in the subject, wherein the presence of both the first andsecond NS3h capture peptides in the mixed biological sample extends thedynamic range for qualitatively detecting the subject antibodiescompared to only using the first or second NS3h capture peptide.
 14. Themethod of claim 13, wherein the biological sample is suspected ofcontaining HCV particles or fragments thereof and the method furthercomprises (e) contacting the sample with a first anti-HCV antibody toform a third complex comprising the first anti-HCV antibody bound to anHCV particle or fragment thereof, (f) contacting the biological samplewith a detectably labeled second anti-HCV antibody that binds the HCVparticle or fragment thereof in the third complex, and (g) detecting thethird complex.
 15. The method of claim 13, further comprising (a)contacting the biological sample with a first HCV core peptide, whereinthe first HCV core peptide specifically binds to at least one of thesubject antibodies in the biological sample to form a fourth complex;and (b) contacting the sample with a second HCV core peptide that isdetectably labeled, wherein the second HCV core peptide binds to thesubject antibody as part of the fourth complex.
 16. The method of claim13, wherein the subject is a human.